Novel MED15::ATF1 fusion in a pediatric melanoma with spitzoid features and aggressive presentation.

Childhood melanoma is a rare and biologically heterogeneous pediatric malignancy. The differential diagnosis of pediatric melanoma is usually broad, including a wide variety of spindle cell or epithelioid neoplasms. Different molecular alterations affecting the MAPK and PI3K/AKT/mTOR pathways, tumor suppressor genes, and telomerase reactivation have been implicated in melanoma tumorigenesis and progression. Here, we report a novel MED15::ATF1 fusion in a pediatric melanoma with spitzoid features and an aggressive clinical course.

Novel HNRNPM::LEUTX fusion resulting from chromothripsis of chromosome 19 in a pediatric undifferentiated small round cell neoplasm.

Small round cell neoplasms comprise a diverse group of tumors characterized by a primitive/undifferentiated appearance. Although several entities are associated with recurrent gene fusions, many of these neoplasms have not been fully characterized, and novel molecular alterations are being discovered. Here, we report an undifferentiated small round cell neoplasm arising in the anterior mediastinum of a 17-month-old female. The tumor harbored a novel HNRNPM::LEUTX fusion resulting from chromothripsis of chromosome 19, which was identified by whole transcriptome sequencing, but not by targeted sequencing. The structural variations caused by the chromothripsis event also challenged the interpretation of the targeted sequencing findings. This report expands the spectrum of gene partners involved in LEUTX fusions and underscores the value of whole transcriptome sequencing in the diagnostic workup of undifferentiated small round cell tumors. It also highlights the interpretive challenges associated with complex genomic alterations. A careful evidence-based analysis of sequencing data along with histopathologic correlation is essential to ensure correct categorization of fusions.

Neonatal osteoblastic tumor with a novel PTBP1::FOSB fusion.

Congenital/neonatal bone neoplasms are extremely rare. We present the case of a patient with a neonatal bone tumor of the fibula that had osteoblastic differentiation and a novel PTBP1::FOSB fusion. FOSB fusions are described in several different tumor types, including osteoid osteoma and osteoblastoma; however, these tumors typically present in the second or third decade of life, with case reports as young as 4 months of age. Our case expands the spectrum of congenital/neonatal bone lesions. The initial radiologic, histologic, and molecular findings supported the decision for close clinical follow-up rather than more aggressive intervention. Since the time of diagnosis, this tumor has undergone radiologic regression without treatment.

A CTNNB1-altered medulloblastoma shows the immunophenotypic, DNA methylation and transcriptomic profiles of SHH-activated, and not WNT-activated, medulloblastoma.

Recent advancements in molecular characterisation have identified four principal molecular groups of medulloblastoma: WNT, SHH, group 3 and group 4. Each has its characteristic clinical features, signature genetic alterations and distinct DNA methylome profiles. Thus far, CTNNB1 mutations have been considered pathognomonic of WNT-activated medulloblastoma. Furthermore, it has been shown that CTNNB1 mutations dominantly drive the WNT-activated phenotype in medulloblastoma, even in the presence of alterations in the SHH pathway. We herein report an illustrative case that challenges this belief-a medulloblastoma with a pathogenic CTNNB1 mutation that otherwise showed the histopathology, immunophenotype and methylation and transcriptomic profiles of an SHH-activated medulloblastoma. Detailed molecular analyses, including whole exome sequencing, transcriptome analysis and DNA methylation profiling with DKFZ brain tumour classifier and St. Jude MLPnet neural network classifier analyses, have been performed on the tumour. Our example emphasises the diagnostic value of the immunohistochemistry panel with YAP1, GAB1 and ß-catenin and DNA methylation profiling, combined with exome sequencing, in the characterisation of medulloblastoma. CTNNB1 mutations are not specific for WNT-activated medulloblastoma, and different CTNNB1 mutations have diverse oncogenic potential.

Host Mesothelin Expression Increases Ovarian Cancer Metastasis in the Peritoneal Microenvironment.

Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLNKO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLNKO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLNWT) or MSLNKO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLNKO mice compared to MSLNWT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.

Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma.

Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors.

Identification of a human papillomavirus-associated oncogenic miRNA panel in human oropharyngeal squamous cell carcinoma validated by bioinformatics analysis of the Cancer Genome Atlas.

High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV(+) OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV(+) OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV(+/-) OPSCC tumors by quantitative PCR-based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy.

Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration.

BACKGROUND: Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. METHODS: We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. RESULTS: Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59% were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30% were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. CONCLUSIONS: We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells.

Progressive metastatic infantile fibrosarcoma with multiple acquired mutations.

Infantile fibrosarcoma is the most common soft-tissue sarcoma in children under the age of 1 yr and is defined molecularly by NTRK fusion proteins. This tumor is known to be locally invasive; however, although rare, metastases can occur. The NTRK fusion acts as a driver for tumor formation, which can be targeted by first- and second-generation TRK inhibitors. Although NTRK gatekeeper mutations have been well-described as mechanisms of resistance to these agents, alternative pathway mutations are rare. Here, we report the case of a patient with infantile fibrosarcoma treated with chemotherapy and TRK inhibition that developed metastatic, progressive disease with multiple acquired mutations, including TP53, SUFU, and an NTRK F617L gatekeeper mutation. Alterations in pathways of SUFU and TP53 have been widely described in the literature in other tumors; however, not yet in infantile fibrosarcoma. Although most patients have a sustained response to TRK inhibitors, a subset will go on to develop mechanisms of resistance that have implications for clinical management, such as in our patient. We hypothesize this constellation of mutations contributed to the patient's aggressive clinical course. Taken together, we report the first case of infantile fibrosarcoma with ETV6::NTRK3 and acquired SUFU, TP53, and NTRK F617L gatekeeper mutation along with detailed clinical course and management. Our report highlights the importance of genomic profiling in recurrent infantile fibrosarcoma to reveal actionable mutations, such as gatekeeper mutations, that can improve patient outcomes.

Kallikrein-5 promotes cleavage of desmoglein-1 and loss of cell-cell cohesion in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) ranks among the top 8 causes of cancer death worldwide, with only a 60% 5-year survival rate, highlighting the need for discovery of novel biomarkers and therapeutic targets. We have previously reported that expression of a panel of serine proteinase kallikreins (KLK 5, 7, 8, and 10) is correlated with formation of more aggressive OSCC tumors in a murine orthotopic OSCC model and is elevated in human OSCC. Current studies focus on understanding the potential role of KLK5 in OSCC progression. In initial studies, KLK levels in malignant OSCC cells (SCC25) were compared with cells from normal oral mucosa (OKF/6) and pre-malignant oral keratinocytes (pp126) using qPCR. A marked elevation of all KLKs was observed in aggressive SCC25 cells relative to OKF/6 cells. In normal skin, KLKs are involved in desquamation during epidermal differentiation via proteolytic cleavage of the desmosomal cadherin component desmoglein 1 (Dsg1). As loss of cell-cell cohesion is prevalent in tumor metastasis, Dsg1 integrity was evaluated. Results show that SCC25 cells exhibit cleavage of Dsg1, which is blocked by proteinase inhibitor treatment as well as by siRNA silencing of KLK5 expression. Furthermore, cell-cell aggregation assays demonstrate that silencing of KLK5 enforces cell-cell adhesion; conversely, overexpression of KLK5 in normal oral mucosal cells (OKF/6) enhances cell dispersal. These data suggest that KLK5 may promote metastatic dissemination of OSCC by promoting loss of junctional integrity through cleavage of desmoglein 1.

Development and evaluation of ActSeq: A targeted next-generation sequencing panel for clinical oncology use.

PURPOSE: The demand for high-throughput genetic profiling of somatic mutations in cancer tissues is growing. We sought to establish a targeted next generation sequencing (NGS) panel test for clinical oncology practice. METHODS: Customized probes were designed to capture exonic regions of 141 genes selected for the panel, which was aimed for the detection of clinically actionable genetic variations in cancer, including KRAS, NRAS, BRAF, ALK, ROS1, KIT and EGFR. The size of entire targeted regions is 0.8 Mb. Library preparation used NEBNext Ultra II FS kit coupled with target enrichment. Paired-end sequencing was run on Illumina NextSeq 500 at a read length of 150 nt. A bioinformatics workflow focusing on single nucleotide variant and short insertions and deletions (SNV/indel) discovery was established using open source, in-house and commercial software tools. Standard reference DNA samples were used in testing the sensitivity and precision and limit of detection in variant calling. RESULTS: The general performance of the panel was observed in pilot runs. Average total reads per sample ranged from 30 million to 48 million, 73% ~82% unique reads. All runs had more than 99% average mapping rate. Mean target coverage ranged from 727x to 879x. Depth of coverage at 50x or more reached 87% of targeted region and 60% of targeted region received 500x or more coverage depth. Using OncoSpan HD827 DNA, which bears 144 variants (SNV/indel) from 80 genes that are within the targeted region on the panel, our somatic variant calling pipeline reached 97% sensitivity and 100% precision respectively, with near 48 million reads. High concordance with orthogonal approaches in variant detection was further verified with 7 cancer cell lines and 45 clinical specimens. CONCLUSION: We developed a NGS panel with a focus on clinically actionable gene mutations and validated the performance in library construction, sequencing and variant calling. High concordance with reference materials and orthogonal mutation detection was observed.

Urinary-type plasminogen activator receptor (uPAR) modulates oral cancer cell behavior with alteration in p130cas.

Oral cavity cancer is among the most frequently diagnosed cancers worldwide and urinary-type plasminogen activator receptor (uPAR) is clinically associated with more invasive tumors and enhanced lymph node metastasis. We seek to further elucidate the mechanism of by which uPAR promotes cell aggressiveness in the unique context of oral squamous cell carcinoma (OSCC). The contribution of uPAR expression to aggressive cellular behavior of OSCC was examined using in vitro cellular models wherein the expression of uPAR was manipulated and in a human OSCC tissue microarray. Results show altered adhesion, motility, and invasion in cells that overexpress uPAR relative to vector control cells. Distinct alterations of focal adhesion protein expression and phosphorylation, including p130cas and paxillin were observed, suggestive of enhanced focal adhesion turnover. Immunohistochemical analysis of microarrayed human OSCC revealed a significant correlation between uPAR and p130cas expression. The non-receptor protein tyrosine kinase c-Src was responsible for the phosphorylation of p130cas in response to uPAR/α3ß1/laminin-5 engagement. Further downstream, the Rho family GTPase Cdc42, but not Rac1, was activated, suggesting a pathway leading to actin reorganization, filopodial protrusion and enhanced motility in uPAR overexpressing oral cancer cells. These data shed light on a molecular mechanism whereby acquisition of uPAR expression may modulate OSCC invasive activity through alteration of focal adhesion dynamics.

In vivo selection of highly metastatic human ovarian cancer sublines reveals role for AMIGO2 in intra-peritoneal metastatic regulation.

The majority of women with ovarian cancer are diagnosed with metastatic disease, therefore elucidating molecular events that contribute to successful metastatic dissemination may identify additional targets for therapeutic intervention and thereby positively impact survival. Using two human high grade serous ovarian cancer cell lines with inactive TP53 and multiple rounds of serial in vivo passaging, we generated sublines with significantly accelerated intra-peritoneal (IP) growth. Comparative analysis of the parental and IP sublines identified a common panel of differentially expressed genes. The most highly differentially expressed gene, upregulated by 60-65-fold in IP-selected sublines, was the type I transmembrane protein AMIGO2. As the role of AMIGO2 in ovarian cancer metastasis remains unexplored, CRISPR/Cas9 was used to reduce AMIGO2 expression, followed by in vitro and in vivo functional analyses. Knockdown of AMIGO2 modified the sphere-forming potential of ovarian cancer cells, reduced adhesion and invasion in vitro, and significantly attenuated IP metastasis. These data highlight AMIGO2 as a new target for a novel anti-metastatic therapeutic approach aimed at blocking cohesion, survival, and adhesion of metastatic tumorspheres.

Urinary-type plasminogen activator (uPA) and its receptor (uPAR) in squamous cell carcinoma of the oral cavity.

OSCC (oral squamous cell carcinoma) is the most common oral malignancy and is estimated to affect approx. 350000 new patients worldwide this year. OSCC is characterized by a high degree of morbidity and mortality, as most patients exhibit local, regional and distant metastasis at the time of diagnosis. Recent genome-wide screening efforts have identified the serine proteinase uPA (urinary-type plasminogen activator, also known as urokinase) as a strong biomarker for prediction of poor disease outcome and a key candidate for molecular classification of oral neoplasms using a 'gene signature' approach. The proteinase uPA binds a surface-anchored receptor designated uPAR (uPA receptor), focalizing proteolytic activity to the pericellular milieu. Furthermore, uPA-uPAR can interact with transmembrane proteins to modify multiple signal transduction pathways and influence a wide variety of cellular behaviours. Correlative clinical data show elevated uPA-uPAR in oral tumour tissues, with tumours exhibiting high levels of both uPA and uPAR as the most invasive. Combined in vitro, pre-clinical and clinical data support the need for further analysis of uPA-uPAR as a prognostic indicator as well as a potential therapeutic target in OSCC.

RNA-seq Reveals the Overexpression of IGSF9 in Endometrial Cancer.

We performed RNA-seq on an Illumina platform for 7 patients with endometrioid endometrial carcinoma for which both tumor tissue and adjacent noncancer tissue were available. A total of 66 genes were differentially expressed with significance level at adjusted p value < 0.01. Using the gene functional classification tool in the NIH DAVID bioinformatics resource, 5 genes were found to be the only enriched group out of that list of genes. The gene IGSF9 was chosen for further characterization with immunohistochemical staining of a larger cohort of human endometrioid carcinoma tissues. The expression level of IGSF9 in cancer cells was significantly higher than that in control glandular cells in paired tissue samples from the same patients (p = 0.008) or in overall comparison between cancer and the control (p = 0.003). IGSF9 expression is higher in patients with myometrium invasion relative to those without invasion (p = 0.015). Reanalysis of RNA-seq dataset from The Cancer Genome Atlas shows higher expression of IGSF9 in endometrial cancer versus normal control and expression was associated with poor prognosis. These results suggest IGSF9 as a new biomarker in endometrial cancer and warrant further studies on its function, mechanism of action, and potential clinical utility.

Aging Increases Susceptibility to Ovarian Cancer Metastasis in Murine Allograft Models and Alters Immune Composition of Peritoneal Adipose Tissue.

Ovarian cancer, the most deadly gynecological malignancy in U.S. women, metastasizes uniquely, spreading through the peritoneal cavity and often generating widespread metastatic sites before diagnosis. The vast majority of ovarian cancer cases occur in women over 40 and the median age at diagnosis is 63. Additionally, elderly women receive poorer prognoses when diagnosed with ovarian cancer. Despite age being a significant risk factor for the development of this cancer, there are little published data which address the impact of aging on ovarian cancer metastasis. Here we report that the aged host is more susceptible to metastatic success using two murine syngeneic allograft models of ovarian cancer metastasis. This age-related increase in metastatic tumor burden corresponds with an increase in tumor infiltrating lymphocytes (TILs) in tumor-bearing mice and alteration of B cell-related pathways in gonadal adipose tissue. Based on this work, further studies elucidating the status of B cell TILs in mouse models of metastasis and human tumors in the context of aging are warranted.

Isolation and identification of eukaryotic initiation factor 4A as a molecular target for the marine natural product Pateamine A.

Natural products continue to demonstrate their utility both as therapeutics and as molecular probes for the discovery and mechanistic deconvolution of various cellular processes. However, this utility is dampened by the inherent difficulties involved in isolating and characterizing new bioactive natural products, in obtaining sufficient quantities of purified compound for further biological studies, and in developing bioactive probes. Key to characterizing the biological activity of natural products is the identification of the molecular target(s) within the cell. The marine sponge-derived natural product Pateamine A (PatA) has been found to be an inhibitor of eukaryotic translation initiation. Herein, we describe the methods utilized for identification of the eukaryotic translation initiation factor 4A (eIF4A) as one of the primary protein targets of PatA. We begin by describing the synthesis of an active biotin conjugate of PatA (B-PatA), made possible by total synthesis, followed by its use for affinity purification of PatA binding proteins from cellular lysates. We have attempted to present the methodology as a general technique for the identification of protein targets for small molecules including natural products.

Primary pigmented nodular adrenocortical disease reveals insulin-like growth factor binding protein-2 regulation by protein kinase A.

OBJECTIVE: Primary pigmented nodular adrenocortical disease (PPNAD) can occur as an isolated trait or part of Carney complex, a familial lentiginosis-multiple endocrine neoplasia syndrome frequently caused by mutations in PRKAR1A, which encodes the 1alpha regulatory subunit of protein kinase A (PKA). Because alterations in the insulin-like growth factor (IGF) axis, particularly IGF-II and IGF binding protein (IGFBP)-2 overexpression, have been implicated in sporadic adrenocortical tumors, we sought to examine the IGF axis in PPNAD. DESIGN: RNA samples and paraffin-embedded sections were procured from adrenalectomy specimens of patients with PPNAD. Changes in expression of IGF axis components were evaluated by real-time quantitative RT-PCR and immunohistochemistry. NCI-H295R cells were used to study PKA and IGF axis signaling in adrenocortical cells in vitro. RESULTS: IGFBP-2 mRNA level distinguished between the two genetic subtypes of this disease; increased IGFBP-2 expression in PRKAR1A mutation-positive PPNAD tissues was also confirmed by immunohistochemistry. Moreover, PKA inhibitors increased IGFBP-2 expression in NCI-H295R adrenocortical cells, and anti-IGFBP-2 antibody reduced their proliferation. CONCLUSIONS: IGFBP-2 expression is increased in PPNAD caused by PRKAR1A mutations, and in adrenocortical cancer cells. This is the first evidence for PKA-dependent regulation of IGFBP-2 expression in adrenocortical cells.