Abdominal Calcifications in a Patient From the Congo Basin with History of Snake Meat Consumption: Radiographic Evidence of Prior Armillifer Infection.

We report a case of a 60-year-old asymptomatic male with history of consumption of uncooked snake meat while living in the Congo basin and prior imaging showing multiple abdominal calcifications. Patient had multiple subepithelial colonic lesions identified during screening colonoscopy and microscopic examination of the lesions demonstrated a calcified nodule in the submucosa with overlying normal mucosa. However, no parasite was identified within the calcified nodule. Given the history of consumption of uncooked snake meat and the typical radiographic feature of multiple abdominal calcifications, it is very likely that the patient's radiographic abnormalities are due to prior Armillifer armillatus infection, a parasitic infection acquired from consumption of uncooked snake meat. Patient was asymptomatic at the time of evaluation and was not given anti-parasitic treatment.

Colonic Ganglioneuroma: A Rare Finding during Colorectal Cancer Screening.

Ganglioneuromas are very rare clinical entities, and their occurrence in the large bowel lays further emphasis on their rarity. Ganglioneuromas are benign tumors of undifferentiated neural crest cells. Their clinical presentation is mostly asymptomatic, and if any symptoms are present at all, they are usually nonspecific, with excellent prognosis. We report an asymptomatic, 65-year-old male with a solitary ascending colonic polyp found on screening colonoscopy. Histology revealed benign polypoid spindle-cell proliferation as well as S100 reactivity, consistent with ganglioneuroma. We report on the clinical presentation and discuss the origin, epidemiology, treatment, and management of this lesion.

An Extremely Rare Case of Advanced Metastatic Small Cell Neuroendocrine Carcinoma of Sinonasal Tract.

Small cell neuroendocrine carcinoma (SNEC) is a rare form of malignancy. It mainly presents as bronchogenic neoplasm, and the extrapulmonary form accounts for only 0.1% to 0.4% of all cancers. These extrapulmonary tumors have been described most frequently in the urinary bladder, prostate, esophagus, stomach, colon and rectum, gall bladder, head and neck, cervix, and skin. Primary SNEC of the sinonasal tract is extremely rare with only less than 100 cases reported in the literature. Because of extreme rarity and aggressiveness of the tumor, the management for this entity varies considerably mandating multimodality approach. In this paper, we report a patient presented with left-sided facial swelling, and the histopathologic examination confirmed primary SNEC of left sinonasal tract. The tumor involved multiple paranasal sinuses with invasion into the left orbit and left infratemporal fossa and metastasized to cervical lymph nodes and bone. The patient encountered devastating outcome in spite of optimal medical management and treatment with palliative chemotherapy highlighting the necessity for further research of primary SNEC of head and neck.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. METHODS: Hepatocellular carcinoma classification defined in our previous transcriptome meta-analysis (S1, S2 and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumours (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumours (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. RESULTS: Hepatocellular carcinoma subclass-predictive indices were steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-foetoprotein (>400 ng/ml) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with the activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184 and 53 to 30, 43 and 22 for S1, S2 and S3 subclass-targeting therapies respectively. CONCLUSIONS: Hepatocellular carcinoma molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.

Spinal cord infarct as a presentation of cholangiocarcinoma with metastases.

It is well-known that malignancies, particularly pancreatic and brain cancers, often present as venous thromboembolism. However, stroke and angina attributable to arterial occlusion are relatively common presentations as well. We are reporting a patient, with treatment-naïve hepatitis C and multiple liver nodules, was admitted for deep vein thrombosis (DVT) and pulmonary embolism (PE). Subsequently, she developed an ascending paralysis due to spinal cord infarct (SCI) despite adequate anticoagulation. She also had an enlargement of left supraclavicular lymph node, which was confirmed histologically metastatic cholangiocarcinoma. To our best knowledge, this is the first literature report showing the association linking SCI to metastatic cholangiocarcinoma as a consequence of hypercoagulable state of malignancy.

Distinguishing nested variants of urothelial carcinoma from benign mimickers by TERT promoter mutation.

Nested variant of urothelial carcinoma (NVUC) is an uncommon variant with minimally atypical cytology, which may overlap with benign urothelial lesions such as von Brunn nests, cystitis cystica, cystitis glandularis, and nephrogenic adenoma. Because of the tumor's deceptively bland appearance, these cancers can potentially be misdiagnosed as benign lesions, leading in some cases to a significant delay in correct diagnosis and appropriate treatment. Prior studies suggest that Ki67 and p53 are useful markers in distinguishing NVUC from benign lesions. However, the overlap in the rates of immunoreactivity has prevented pathologists from using these markers as reliable adjunct markers in differentiating NVUC from mimickers. In addition, large nested variant urothelial carcinoma (LNVUC), a relatively new entity, shares features of both the NVUC and papillary urothelial carcinomas with an inverted growth pattern. They also mimic benign lesions, such as proliferation of von Brunn nests and inverted urothelial papilloma. With the recent demonstration of a strong association of TERT promoter mutations and urothelial carcinoma, we hypothesized that TERT promoter mutations would be a useful marker to distinguish NVUC and LNVUC from other benign urothelial lesions. We have therefore sequenced the TERT promoter region of 20 cases of NVUC, 10 cases of LNVUC, 5 cases of von Brunn nests, 3 cases of cystitis cystica, 3 cases of cystitis glandularis, and 3 cases of nephrogenic adenoma. We found that 17 of 20 cases of NVUC and 8 of 10 cases of LNVUC had TERT promoter mutation: C228T; no mutation was found in any of the benign mimickers (0/14). This result strongly suggests that TERT promoter mutation is a useful adjunct biomarker to distinguish NVUC and LNVUC from benign mimickers.

Molecular, histopathological, and genomic variants of glioblastoma.

Glioblastoma (GBM), the most common primary brain tumor, has a poor median prognosis despite modern surgical, chemotherapeutic, and radiation modalities, which have shown little clinical efficacy. Initially categorized by clinicopathological classification into de novo primary GBM and secondary GBM, which arises from lower-grade glioma, genomic studies have elucidated several distinct genotypes. In addition, distinct patterns of dysregulated epidermal growth factor receptor, platelet-derived growth factor receptor, p53, phosphatase and tensin homolog, cell cycle proteins, and isocitrate dehydrogenase 1, as well as loss of heterozygosity in multiple chromosomes complicate the GBM mutational landscape. Even with the many approaches in targeting these mutations, a long-standing clinical cure remains limited because of the tremendous heterogeneity and challenges in developing targeted treatments. Furthermore, this cancer utilizes ingenious approaches to subvert targeted agents and pathological variants of GBM demonstrate distinct molecular signatures, which may impact prognosis. This review discusses the collective understanding of GBM heterogeneity, including molecular, histopathological, and genomic features; why treatments have failed in the past; and how future clinical trials and therapies can be devised.

CTX-M ß-lactamase-producing Klebsiella pneumoniae in suburban New York City, New York, USA.

CTX-M extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolates are infrequently reported in the United States. In this study, we analyzed nonduplicate ESBL-producing K. pneumoniae and Escherichia coli clinical isolates collected during 2005-2012 at a tertiary care medical center in suburban New York City, USA, for the presence of blaCTX-M, blaSHV, blaTEM, and blaKPC genes. Despite a high prevalence of blaCTX-M genes in ESBL-producing E. coli since 2005, blaCTX-M genes were not detected in K. pneumoniae until 2009. The prevalence of CTX-M-producing K. pneumoniae increased significantly over time from 1.7% during 2005-2009 to 26.4% during 2010-2012 (p<0.0001). CTX-M-15 was the dominant CTX-M genotype. Pulsed-field gel electrophoresis and multilocus sequence typing revealed high genetic heterogeneities in CTX-M-producing K. pneumoniae isolates. This study demonstrates the recent emergence and polyclonal spread of multidrug resistant CTX-M-producing K. pneumoniae isolates among patients in a hospital setting in the United States.

Sensitive detection of BRAF V600E mutation by Amplification Refractory Mutation System (ARMS)-PCR.

BACKGROUND: BRAF mutations occur in approximately 8% of all human cancers and approach 50% in melanoma and papillary carcinoma of thyroid. These mutations provide potentially valuable diagnostic, prognostic and treatment response prediction markers. A sensitive, specific, low-cost assay to detect these mutations is needed. RESULTS: To detect BRAF V600E mutation in formalin-fixed, paraffin-embedded (FFPE) tissue, we developed a method using Amplification Refractory Mutation System (ARMS)-PCR. This method was designed to amplify three products in a single reaction tube: a 200 bp common product serving as an amplification control, a 144 bp BRAF V600E specific product, and a 97 bp wild-type (wt) specific product. The sensitivity of this method was determined to be as low as 0.5% for the BRAF V600E allele in a wild-type background. This method was successfully validated in 72 thyroid tumors. It detected V600E mutation in 22 out of 33 (67%) of the conventional papillary thyroid carcinoma (PTC), 8 out of 12 (75%) of the tall-cell variant of PTC, whereas none of the 10 follicular variant of PTC showed BRAF V600E mutation. In addition, none of the 14 follicular adenomas and 3 follicular carcinomas had BRAF V600E mutation. As a comparison method, direct dideoxy sequencing found only 27 out of 30 (90%) mutations detected by ARMS-PCR method, suggesting that this ARMS-PCR method has higher sensitivity. CONCLUSIONS: Our ARMS-PCR method provides a new tool for rapid detection of BRAF V600E mutation. Our results indicate that ARMS-PCR is more sensitive than automated dideoxy sequencing in detecting low BRAF V600E allele burdens in FFPE tumor specimen. The strategy of this ARMS-PCR design may be adapted for early detection of point mutations of a variety of biomarker genes.

Current understanding of the role and targeting of tumor suppressor p53 in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common primary malignancy in the brain and confers a uniformly poor prognosis. Despite decades of research on the topic, limited progress has been made to improve the poor survival associated with this disease. GBM arises de novo (primary GBM) or via dedifferentiation of lower grade glioma (secondary GBM). While distinct mutations are predominant in each subtype, alterations of tumor suppressor p53 are the most common, seen in 25-30 % of primary GBM and 60-70 % of secondary GBM. Various roles of p53 that protect against neoplastic transformation include modulation of cell cycle, DNA repair, apoptosis, senescence, angiogenesis, and metabolism, resulting in an extremely complex signaling network. Mutations of p53 in GBM are most common in the DNA-binding domain, namely within six hotspot mutation sites (codons 175, 245, 248, 249, 273, and 282). These alterations generally result in loss-of-function, gain-of-function, and dominant-negative mutational effects for p53, however, the distinct effect of these mutation types in GBM pathogenesis remain unclear. Signaling alterations downstream from p53 (e.g., MDM2, MDM4, INK4/ARF), p53 isoforms (e.g., p63, p73), and microRNAs (e.g., miR-34) also play critical roles in modulating the p53 pathway. Despite novel mouse models of GBM showing that p53 combined with other mutation generate tumors de novo, the role of p53 as a molecular marker of GBM remains controversial with most studies failing to show an association with prognosis. Regarding treatment in GBM, p53 targeted-gene therapy and vaccinations have reached phase I clinical trials while therapeutic drugs are still in preclinical development. This review aims to discuss the most recent findings regarding the impact of p53 mutations on GBM pathogenesis, prognosis, and treatment.

B-Raf and the inhibitors: from bench to bedside.

The B-Raf protein is a key signaling molecule in the mitogen activated protein kinase (MAPK) signaling pathway and has been implicated in the pathogenesis of a variety of cancers. An important V600E mutation has been identified and can cause constitutive B-Raf activation. Recent studies have evaluated a variety of small molecule inhibitors targeting B-Raf, including PLX4032/vemurafenib, dabrafenib, LGX818, GDC0879, XL281, ARQ736, PLX3603 (RO5212054), and RAF265. Therapeutic resistance has been identified and various mechanisms described. This review also discussed the current understanding of B-Raf signaling mechanism, methods of mutation detection, treatment strategies as well as potential methods of overcoming therapeutic resistance.

A functional recombinant human 4-1BB ligand for immune costimulatory therapy of cancer.

Costimulatory factors hold great promise for development into novel anticancer biotherapeutics. An agonist to 4-1BB is ranked number 8 by National Cancer Institute on the list of 20 agents with high potential for use in treating cancer. We earlier reported on a recombinant murine 4-1BB ligand fusion protein that binds 4-1BB receptor on murine T cells and stimulates their proliferation in tumor-bearing mice. To facilitate clinical translation,we constructed a corresponding recombinant human 4-1BB ligand fusion protein (hIg-h4-1BBLs) and showed its ability to activate human T cells in vitro. Using Chinese hamster ovary cells transformed with a plasmid coexpressing hIg-h4-1BBLs and rat glutamine synthetase, we generated a high-producing clone by sequential selection with methionine sulfoximine. The hIg-h4-1BBLs was partially purified by protein A column chromatography and characterized biochemically and functionally, using human 4-1BB binding and human T-cell proliferation assays, in vitro.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot confirmed that the hIg-h4-1BBLs is expressed predominantly as a functionally active multimeric protein with the ability to specifically bind to cells expressing human 4-1BB receptor and induce significant T-cell proliferation in vitro using both human and monkey peripheral blood mononuclear cells. The hIg-h4-1BBLs can be produced in large quantities from the high producer clone and developed as a novel immune costimulatory biotherapeutic to treat, alone and in combination with other modalities, various malignant diseases in patients through T-cell activation. Process development of this clinical agent has been discussed with the Food and Drug Administration in a pre-Investigational New Drug meeting and presented to the Office of Biotechnology Activities in a public hearing.

The administration of IL-12/GM-CSF and Ig-4-1BB ligand markedly decreases murine floor of mouth squamous cell cancer.

OBJECTIVE: To assess immune-based gene therapy in a murine floor of mouth (FOM) squamous cell carcinoma (SCC) model. STUDY DESIGN: In vitro and in vivo testing of immune therapy for SCC. METHODS: Multiple SCC lines were infected by using advRSV-interleukin-12 (IL-12) and advCMV-interleukin-12/granulocyte macrophage colony-stimulating factor (IL-12/GM-CSF) and monitored for production of IL-12 and GM-CSF. Intratumoral injections of viral vectors were administered with systemic Ig-4-1BB ligand in an orthotopic murine FOM SCC model and followed for tumor size and survival. RESULTS: In vitro, all cell lines produced substantial levels of IL-12 and GM-CSF. In vivo, tumors treated with advCMV-IL-12/GM-CSF and Ig-4-1BBL showed a striking reduction in tumor volume (vs control P<0.0001) and improved median survival (38 days vs 19 days for control, P<0.0001). CONCLUSION: Combination immune-based therapies effectively improve survival in mice bearing FOM SCC over single-modality therapy.

rVSV(M Delta 51)-M3 is an effective and safe oncolytic virus for cancer therapy.

Oncolytic vesicular stomatitis virus (VSV) is being developed as a novel therapeutic agent for cancer treatment, although it is toxic in animals when administered systemically at high doses. Its safety can be substantively improved by an M Delta 51 deletion in the viral genome, and yet VSV(M Delta 51) induces a much greater, robust cellular inflammatory response in the host than wild-type VSV, which severely attenuates its oncolytic potency. We have reported that the oncolytic potency of wild-type VSV can be enhanced by vector-mediated expression of a heterologous viral gene that suppresses cellular inflammatory responses in the lesions. To develop an effective and safe VSV vector for cancer treatment, we tested the hypothesis that the oncolytic potency of VSV(M Delta 51) can be substantively elevated by vector-mediated expression of M3, a broad-spectrum and high-affinity chemokine-binding protein from murine gammaherpesvirus-68. The recombinant vector rVSV(M Delta 51)-M3 was used to treat rats bearing multifocal lesions (1-10 mm in diameter) of hepatocellular carcinoma (HCC) in their liver by hepatic artery infusion. Treatment led to a significant reduction of neutrophil and natural killer cell accumulation in the lesions, a 2-log elevation of intratumoral viral titer, substantively enhanced tumor necrosis, and prolonged animal survival with a 50% cure rate. Importantly, there were no apparent systemic and organ toxicities in the treated animals. These results indicate that the robust cellular inflammatory responses induced by VSV(M Delta 51) in HCC lesions can be overcome by vector-mediated intratumoral M3 expression, and that rVSV(M Delta 51)-M3 can be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future.

Rejection of metastatic 4T1 breast cancer by attenuation of Treg cells in combination with immune stimulation.

4T1 breast carcinoma is a highly malignant and poorly immunogenic murine tumor model that resembles advanced breast cancer in humans, and is refractory to most immune stimulation-based treatments. We hypothesize that the ineffectiveness of immune stimulatory treatment is mediated by the suppressive effects of CD4(+)CD25(+) regulatory T (Treg) cells, which can be attenuated by engaging the glucocorticoid-induced tumor necrosis factor receptor family-related protein with its natural ligand (GITRL); further, combination treatment with existing immune stimulation regimens will augment anti-tumor immunity and eradicate metastatic 4T1 tumors in mice.A soluble homodimeric form of mouse GITRL (mIg-mGITRLs) was molecularly constructed and used to treat orthotopic 4T1 tumors established in immune-competent, syngeneic Balb/c mice. When applied in combination with adenovirus-mediated intratumoral murine granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) gene delivery plus systemic 4-1BB activation, mIg-mGITRLs attenuated the immune-suppressive function of splenic Treg cells, which led to elevated interferon-gamma (IFN-gamma) production, tumor-specific cytolytic T-cell activities, tumor rejection and long-term survival in 65% of the animals without apparent toxicities. The results demonstrate that addition of mIg-mGITRLs to an immune-stimulatory treatment regimen significantly improved long-term survival without apparent toxicity, and could potentially be clinically translated into an effective and safe treatment modality for metastatic breast cancer in patients.

Oncolysis of hepatic metastasis of colorectal cancer by recombinant vesicular stomatitis virus in immune-competent mice.

With currently available treatments, patients with metastatic colorectal cancer (CRC) have a median survival of 14.8 months and a 5-year survival rate of less than 10%. In recent years, tumor-targeted replicating viruses have rapidly emerged as potential novel oncolytic agents for cancer treatment. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with inherent selectivity for replication in tumor cells due to their attenuated antiviral response. VSV is particularly appealing as an oncolytic agent for its exceptionally rapid replication cycle in tumor cells, whereby it is capable of manifesting its maximal oncolytic effects before the onset of neutralizing antiviral immune responses in the host. In this study, we used a recombinant VSV vector expressing the green fluorescent protein gene (rVSV-GFP) to monitor VSV replication easily in CRC cells. Using this GFP-expressing virus, we found that rVSV-GFP efficiently replicated and lysed murine and human CRC cell lines in vitro. We also evaluated the potential of rVSV-GFP to treat MCA26 CRC metastases implanted orthotopically into the livers of syngeneic BALB/c mice. We provide conclusive evidence that rVSV-GFP is able to replicate extensively in the tumors, but not in normal liver cells, in tumor-bearing mice. A single intratumoral injection also caused extensive tumor necrosis, which led to a significant prolongation of animal survival. Our results indicate that VSV can be an effective and safe oncolytic agent against hepatic CRC metastasis in immune-competent mice and may be developed for the treatment of cancer patients in the future.

Oncolytic vesicular stomatitis virus for treatment of orthotopic hepatocellular carcinoma in immune-competent rats.

Tumor-targeted replicating viruses are being developed as a novel class of oncolytic agents. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with inherent specificity for replication in tumor cells due to their attenuated antiviral responses. VSV as an oncolytic virus is particularly appealing for its exceptionally rapid replication rate in tumor cells, such that the oncolytic effects could be maximally manifested before the onset of potentially neutralizing antiviral immune responses in the host. To easily monitor VSV replication, we have rescued a recombinant VSV (rVSV) vector expressing the green fluorescent protein (GFP) gene (rVSV-GFP). Using this GFP-expressing virus, we have demonstrated the oncolytic potential of VSV against human and rat hepatocellular carcinoma (HCC). We found that rVSV-GFP replicated efficiently in cultured human and rat HCC cells, whereas normal human and rat hepatocytes were refractory. When a single dose of the vector was injected intratumorally into large orthotopically implanted HCC in immune-competent rats, rVSV-GFP effectively and selectively replicated throughout the solid tumor mass without apparent hepatotoxicity, caused tumor destruction, and inhibited tumor growth, which led to significant prolongation of animal survival. Our results show that VSV is an effective oncolytic agent against HCC in immune-competent hosts and warrants further development for future therapy in patients with HCC.