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Apart from autopsy, tissue correlates of coronavirus disease 2019 (COVID-19) clinical stage are lacking. In the current study, cutaneous punch biopsy specimens of 15 individuals with severe/critical COVID-19 and six with mild/moderate COVID-19 were examined. Evidence for arterial and venous microthrombi, deposition of C5b-9 and MASP2 (representative of alternative and lectin complement pathways, respectively), and differential expression of interferon type I-driven antiviral protein MxA (myxovirus resistance A) versus SIN3A, a promoter of interferon type I-based proinflammatory signaling, were assessed. Control subjects included nine patients with sepsis-related acute respiratory distress syndrome (ARDS) and/or acute kidney injury (AKI) pre-COVID-19. Microthrombi were detected in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P 0.001). Cells lining the microvasculature staining for spike protein of severe acute respiratory syndrome coronavirus 2, the etiologic agent of COVID-19, also expressed tissue factor. C5b-9 deposition occurred in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P 0.001). MASP2 deposition was also restricted to severe/critical COVID-19 cases. MxA expression occurred in all six mild/moderate versus two (15%) of 13 severe/critical cases (P 0.001) of COVID-19. In contrast, SIN3A was restricted to severe/critical COVID-19 cases co-localizing with severe acute respiratory syndrome coronavirus 2 spike protein. SIN3A was also elevated in plasma of patients with severe/critical COVID-19 versus control subjects (P ≤ 0.02). In conclusion, the study identified premortem tissue correlates of COVID-19 clinical stage using skin. If validated in a longitudinal cohort, this approach could identify individuals at risk for disease progression and enable targeted interventions.

Purpose:Pelareorep is an intravenously delivered oncolytic reovirus that can induce a T-cell–inflamed phenotype in pancreatic ductal adenocarcinoma (PDAC). Tumor tissues from patients treated with pelareorep have shown reovirus replication, T-cell infiltration, and upregulation of PD-L1. We hypothesized that pelareorep in combination with pembrolizumab and chemotherapy in patients with PDAC would be safe and effective.Patients and Methods:A phase Ib single-arm study enrolled patients with PDAC who progressed after first-line treatment. Patients received pelareorep, pembrolizumab, and either 5-fluorouracil, gemcitabine, or irinotecan until disease progression or unacceptable toxicity. Study objectives included safety and dose-limiting toxicities, tumor response, evaluation for reovirus replication, and immune analysis in peripheral blood and tumor biopsies.Results:Eleven patients were enrolled. Disease control was achieved in three of the 10 efficacy-evaluable patients. One patient achieved partial response for 17.4 months. Two additional patients achieved stable disease, lasting 9 and 4 months, respectively. Treatment was well tolerated, with mostly grade 1 or 2 treatment-related adverse events, including flu-like symptoms. Viral replication was observed in on-treatment tumor biopsies. T-cell receptor sequencing from peripheral blood revealed the creation of new T-cell clones during treatment. High peripheral clonality and changes in the expression of immune genes were observed in patients with clinical benefit.Conclusions:Pelareorep and pembrolizumab added to chemotherapy did not add significant toxicity and showed encouraging efficacy. Further evaluation of pelareorep and anti–PD-1 therapy is ongoing in follow-up studies. This research highlights the potential utility of several pretreatment and on-treatment biomarkers for pelareorep therapy warranting further investigation.

Dosing schedule figure Dose reduction table

Purpose: Primary central nervous system posttransplant lymphoproliferative disorder (PCNS-PTLD) is a complication of solid organ transplantation with a poor prognosis and typically associated with Epstein–Barr virus (EBV). We hypothesized EBV lytic-phase protein expression would allow successful treatment with antiviral therapy.Patients and Methods: Thirteen patients were treated with zidovudine (AZT), ganciclovir (GCV), dexamethasone, and rituximab in EBV+ PCNS-PTLD. Twice-daily, intravenous AZT 1,500 mg, GCV 5 mg/kg, and dexamethasone 10 mg were given for 14 days. Weekly rituximab 375 mg/m2 was delivered for the first 4 weeks. Twice-daily valganciclovir 450 mg and AZT 300 mg started day 15. Lytic and latent protein expression was assessed using in situ hybridization and immunohistochemistry. Immunoblot assay assessed lytic gene activation. Cells transfected with lytic kinase vectors were assessed for sensitivity to our therapy using MTS tetrazolium and flow cytometry.Results: The median time to response was 2 months. Median therapy duration was 26.5 months. Median follow-up was 52 months. The estimated 2-year overall survival (OS) was 76.9% (95% CI, 44.2%–91.9%). Overall response rate (ORR) was 92% (95% CI, 64%–100%). BXLF1/vTK and BGLF4 expression was found in the seven tumor biopsies evaluated. Lytic gene expression was induced in vitro using the four-drug regimen. Transfection with viral kinase cDNA increased cellular sensitivity to antiviral therapy.Conclusions: EBV+ PCNS-PTLD expressed lytic kinases and therapy with AZT, GCV, rituximab and dexamethasone provided durable responses. Induction of the lytic protein expression and increased cellular sensitivity to antiviral therapy after transfection with viral kinase cDNA provides a mechanistic rationale for our approach. Clin Cancer Res; 24(14); 3273–81. ©2018 AACR.

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Purpose: Hepatocellular cancer (HCC) is highly resistant to chemotherapy and is associated with poor prognosis. Chronic hepatitis C virus (HCV) infection is a major cause of HCC. However, the effect of viral proteins in mediating chemosensitivity in tumor cells is unknown. We postulated that HCV viral proteins could modulate therapeutic responses by altering host cell microRNA (miRNA) expression.Experimental Design: HepG2 malignant hepatocytes were stably transfected with full-length HCV genome (Hep-394) or an empty vector (Hep-SWX). MiRNA profiling was done by using a custom microarray, and the expression of selected miRNAs was validated by real-time PCR. Protein expression was assessed by Western blotting, whereas caspase activation was assessed by a luminometric assay.Results: The IC50 to sorafenib was lower in Hep-394 compared with Hep-SWX control cells. Alterations in miRNA expression occurred with 10 miRNAs downregulated >2-fold and 23 miRNAs upregulated >2-fold in Hep-394 cells compared with controls. Of these, miR-193b was overexpressed by 5-fold in Hep-394 cells. miR-193b was predicted to target Mcl-1, an antiapoptotic protein that can modulate the response to sorafenib. The expression of Mcl-1 was decreased, and basal caspase-3/7 activity and poly ADP ribose polymerase cleavage were increased in Hep-394 cells compared with controls. Moreover, transfection with precursors to miR-193b decreased both Mcl-1 expression and the IC50 to sorafenib.Conclusions: Cellular expression of full-length HCV increases sensitivity to sorafenib by the miRNA-dependent modulation of Mcl-1 and apoptosis. Modulation of miRNA responses may be a useful strategy to enhance response to chemotherapy in HCC. Clin Cancer Res; 16(3); 957–66

PDF file - 636KB, Supplemental Table S1: information of the GBM lines used in this paper. Supplemental Table S2: information of the patient clinical data for Figure 1. Supplemental Table S3: Percentage of cells positive for staining with PRMT5 in spontaneous high grade astrocytoma from Mut3 strain (GFAP-cre; cisNf1-/+; P53-/-) and normal brain tissue from wild-type control mouse stained by PRMT5 and ki-67 antibody. Supplemental table S4. GBM cell line (U251) was transfected by scramble control or si-PRMT5. Affymetrix microarray genechip was performed in three independent experiments to evaluate genes up-regulated with PRMT5 silencing.

PDF file - 1690KB, Supplemental Figure S1. PRMT5 is over-expressed in human GBM cell lines. Supplemental Figure S2: High grade astrocytomas Supplemental Figure S3: efficacy and specificity of PRMT5 knock-down by lead siRNA. Supplemental Figure S4: PRMT5 Knockdown promotes cell death of human GBM cells Supplemental Figure S5: si-PRMT5 induced cell death is P53-independent Supplemental Figure S6: Decreased ST7 expression correlates with worse survival in all glioma patients (grade I-IV). Supplemental Figure S7: Over-expression of ST7 in three GBM cell lines was confirmed by real-time PCR and western blot (top panels).

Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease. Cancer Res; 74(6); 1752–65. ©2014 AACR.

PDF file - 596K, Table S1: Predicted GSK3b phosphorylation sites on SMO. TableS2: Predicted GSK3b phosphorylation sites on SMO. FIG S1: Generation of cell lines resistant to increasing concentration of tamoxifen. FIG S2: Mammosphere formation assay with cells resistant to increasing concentration of tamoxifen. FIG S3: Effect of Gli1 depletion on OHTR and T47D cells. FIG S4: Regulation of GLI1 by PI3k/Akt pathway.

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand their role in TKI-resistant NSCLCs, we examined changes in miRNA that are mediated by tyrosine kinase receptors. Here we report that miR-30b, miR-30c, miR-221 and miR-222 are modulated by both epidermal growth factor (EGF) and MET receptors, whereas miR-103 and miR-203 are controlled only by MET. We showed that these miRNAs have important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition of NSCLC cells in vitro and in vivo by inhibiting the expression of the genes encoding BCL2-like 11 (BIM), apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ɛ (PKC-ɛ) and sarcoma viral oncogene homolog (SRC). These findings suggest that modulation of specific miRNAs may provide a therapeutic approach for the treatment of NSCLCs.

An 11-year-old female presented with multiple oral lesions for several months. Histopathological findings suggested focal epithelial hyperplasia (FEH), also known as Heck disease. FEH is strongly associated with Human papillomavirus (HPV), especially genotypes 13 and 32. An oral swab of a mucosal lesion was subsequently obtained for cytology, immunohistochemistry and in situ hybridization. In addition, in situ hybridization and immunohistochemistry were also performed retrospectively on the biopsy specimen for correlation. The cytology specimen showed squamous cells with enlarged, slightly atypical nuclei and rare perinuclear halos. The histology findings included papillomatosis with acanthosis, mild nuclear atypia and focal perinuclear halos. The immunohistochemistry for the consensus HPV L1 capsid protein was found in both the cytology and biopsy specimens indicating that the lesion was HPV-related. High viral copy numbers of HPV 13 were detected by in situ hybridization in both the cytology and histology specimens. Although histologic features of FEH have been well characterized in the literature, to our knowledge, this is the first case to describe in FEH with adjunct immunohistochemistry and in situ hybridization results. Furthermore, these findings assisted in our diagnosis since the patient's clinical presentation was a diagnostic challenge with smooth dome-shaped papules instead of the typically described flat-topped verrucous lesions seen in FEH. In summary, our case reveals that there is a high concordance between the HPV 13 detection in the cytology and histology of FEH, and that performing cytology in addition to histology can be used to optimize diagnostic evaluation towards appropriate patient care.

Human papilloma virus (HPV) is the causative agent for a variety of cutaneous lesions including verruca vulgaris (VV) and epidermodysplasia verruciformis (EDV). There are more than 200 known genotypes of HPV, and specific HPV types are associated with different clinical manifestations and malignant potentials. Herein, we describe a case of a 43-year-old immunocompetent woman who presented with morphologically distinct lesions that were most consistent with EDV on clinical examination. However, further histopathological and viral analysis confirmed the lesions as HPV-57-positive VV. The risk of malignant transformation, and therefore treatment and surveillance, is dramatically different in VV versus EDV. Therefore, this case highlights the importance of a proper histopathological diagnosis with HPV viral testing when clinical presentations may vary or mimic other diseases.

Reovirus is an oncolytic virus with natural tropism for cancer cells. We previously showed that reovirus intravenous administration in myeloma patients was safe, but disease control associated with viral replication in the cancer cells was not observed. Here we show that ex vivo proteasome inhibitors (PIs) potentiate reovirus replication in circulating classical monocytes, increasing viral delivery to myeloma cells. We found that the anti-viral signals in monocytes primarily rely on the NF-kB activation and that this effect is impaired by the addition of PIs. Conversely, PIs improved reovirus-induced monocyte and T cell activation against cancer cells. Based on these preclinical data, we conducted a phase 1b trial of the reovirus Pelareorep together with the PI carfilzomib in 13 heavily pretreated bortezomib-resistant MM patients. Objective responses associated with reovirus active replication in MM cells, T cell activation and monocytic expansion were noted in 70% of patients.

Vascular leak is a major sequela of inflammation, which is associated with arrhythmic pathologies such as atrial fibrillation (AF) and myocardial infarction (MI). We recently demonstrated that the vascular leak-inducing cytokine vascular endothelial growth factor (VEGF; 90-580 pg/ml - levels found in AF patients) induces acute remodeling (30-60 minutes) of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains, disrupting their ultrastructure and prompting translocation of Na V 1.5 from these sites. This in turn disrupted impulse propagation and promoted arrhythmias in murine atria. Here, we tested the hypotheses that i) similar acute pro-arrhythmic remodeling occurs in the ventricles of MI patients, and ii) protecting the vascular barrier may prevent arrhythmias following an acute inflammatory insult. First, we examined myocardial samples from five human MI patients. VEGF was overexpressed in both cardiomyocytes and vascular endothelium in the border zone surrounding V 1.5 near both connexin43 and N-cadherin within the border zone in 1-, 3-, and 9-day-old infarcts, paralleling our observations in mouse atria. Next, we returned to our murine model of AF induced by acute inflammatory insult (100 pg/ml VEGF for 60 minutes) to test the antiarrhythmic efficacy of protecting the vascular endothelial barrier. Overall, median in vivo arrhythmia burden was higher in VEGF-treated mice relative to vehicle controls (7.5±11 vs. 0±6 s/hr). We tested two strategies shown to prevent vascular barrier breakdown: Blocking connexin43 hemichannels (αCT11 peptide) decreased in vivo arrhythmia burden to 0 ± 6.07 s/hr. Panx1-IL2 (a peptide inhibitor of Panx1 channels) treatment decreased also in vivo arrhythmia burden (0 ± 15.57 s/hr with 1.6 μM Panx1-IL2). Similar antiarrhythmic efficacy was also achieved with small molecule inhibitors of Cx43 and Panx1. These results highlight VEGF-induced vascular leak as a novel mechanism for acute arrhythmias both in the early stage AF and following MI. Indeed, this mechanism may contribute to post-MI AF. Importantly, vascular-barrier protection may be a viable strategy to prevent these arrhythmias.

Lung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs, frequently deregulated in human malignancies. We now report that miR221&222 are over-expressed in aggressive non small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation, through the c-Jun transcription factor.

Aortic aneurism open repair surgery can cause spinal cord (SC) injury with 5-15% of patients developing paraparesis or paraplegia. Using a mouse model of transient aortic cross-clamping (ACC), we have previously found that the expression of proinflammatory microRNA

Objective Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. Design and results Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein–Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. Conclusions We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.

Objective—To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses. Sample—Laminae from the forelimb hooves of 8 healthy horses. Procedures—Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization. Results—Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae. Conclusions and Clinical Relevance—Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.

283 Background: Pelareorep (REOLYSIN) is a immuno-oncolytic virus (IOV) that induces an inflamed tumor phenotype secondary to viral infection of cancer cells. In combination with chemotherapy, it achieves 1 & 2 year-survival rates of 46% & 24% in MAP pts, respectively. Tumor analysis from pts showed reovirus protein replication, T-cell infiltration and upregulation of PD-L1. Similarly, the combination of pelareorep with anti-PD-1 antibody documented survival benefit in a pre-clinical model. We hypothesized that pelareorep in combination with chemo and pembrolizumab in pts with MAP would be clinically efficacious. Methods: A phase 1b study enrolled MAP pts who progressed after first line treatment. Pts received pelareorep (4.5 x 10 10TCID 50 IV, D1 & D2), plus pembrolizumab (2mg/kg IV, D8) plus either 1)5-FU (LV (200 mg/m2 /5-FU 200 mg /m2 IV bolus, 5-FU 1200mg/m2 continuous IV infusion D1) or 2) gemcitabine (1000 mg/m2 IV, D1), or 3) irinotecan (125 mg/m2 IV, D1) q3w, until disease progression/unacceptable toxicity. The primary endpoint was safety. Secondary objectives included tumor response & evaluation for reovirus replication/immune analysis. Results: 11 pts were enrolled with pelareorep, pembrolizumab and gem (n = 6), 5-FU (n = 3), or iri (n = 2). Most common grade 1 or 2 TEAEs include: fever (73%), headache (55%), chills (46%), dehydration (36%), fatigue (27%) and anemia (27%). One pt (gem arm), transient Gr 2 increased transaminases was reported on two occasions. Grade 3 or 4 TEAEs occurred in 8 pts (73%): abdominal pain, anemia, arthralgias, biliary obstruction, chills, DVT, diarrhea, fever, hyperglycemia, leukopenia, myalgias, nausea, neutropenia, pulmonary emboli, urinary tract infection and vomiting. Of the 5 efficacy evaluable pts, one had PR (13.8 m duration) and 2 SD (lasting 126 and 277 days). Eight died secondary to PD. On-treatment biopsy show reovirus infection in cancer cells and immune infiltrates. Conclusions: The combination therapy showed manageable safety profiles and antitumor activity in previously treated MAP pts. Further evaluation of anti-tumor activity of pelareorep and anti-PD-1 antibody ± chemotherapy combos is planned. Clinical trial information: NCT02620423.

Background The purpose of this study is to determine whether microRNA for pluripotent stem cells are also expressed in breast cancer and are associated with metastasis and outcome. Methods We studied global microRNA profiles during differentiation of human embryonic stem cells (n =26) and in breast cancer patients (n = 33) and human cell lines (n = 35). Using in situ hybridization, we then investigated MIR302 expression in 318 untreated breast cancer patients (test cohort, n = 22 and validation cohort, n = 296). In parallel, using next-generation sequencing data from breast cancer patients (n = 684), we assessed microRNA association with stem cell markers. All statistical tests were two-sided. Results In healthy tissues, the MIR302 (high)/MIR203 (low) asymmetry was exclusive for pluripotent stem cells. MIR302 was expressed in a small population of cancer cells within invasive ductal carcinoma, but not in normal breast (P < .001). Furthermore, MIR302 was expressed in the tumor cells together with stem cell markers, such as CD44 and BMI1. Conversely, MIR203 expression in 684 breast tumors negatively correlated with CD44 (Spearman correlation, Rho = -0.08, P = .04) and BMI1 (Rho = -0.11, P = .004), but positively correlated with differentiation marker CD24 (Rho = 0.15, P < .001). Primary tumors with lymph node metastasis had cancer cells showing scattered expression of MIR302 and widespread repression of MIR203. Finally, overall survival was statistically significantly shorter in patients with MIR302-positive cancer cells (P = .03). Conclusions In healthy tissues the MIR302(high)/MIR203(low) asymmetry was characteristic of embryonic and induced pluripotency. In invasive ductal carcinoma, the MIR302/MIR203 asymmetry was associated with stem cell markers, metastasis, and shorter survival.

A 73-year-old woman developed multiple depigmented lesions in the fundus 4-6 months after an episode of acute Herpes zoster ophthalmicus. Post-mortem examination of the globe 15 years after this acute episode confirmed multiple old chorioretinal scars probably due to vasculitis of the short posterior ciliary arteries and branches. Patchy old infarcts were also noted in the iris.

Human CD4, the receptor for the gp120 envelope glycoprotein of HIV-1, is the route for viral entry into CD4+ cells; other cellular factors may cooperate with CD4 to facilitate HIV-1 entry into human cells. Human CD4 expressed on murine cells does not readily mediate HIV-1 entry, which may reflect a functional incompatibility of human CD4 with murine cellular components. We postulated that a HIV-1 gp120-binding mutant murine CD4 (L3T4) possessing a minimal number of human amino acid residues could facilitate HIV-1 entry into rodent cells, unlike human CD4. This hypothesis led us to develop a series of murine L3T4 mutants that bear human CD4 gp120-binding region amino acid residues while retaining most L3T4 epitopes. HeLa cell transfectants expressing gp120-binding mutant L3T4 proteins could be infected with HIV-1. Three mouse cell lines expressing these L3T4 mutant proteins could also be infected with HIV-1 as determined by PCR techniques that detect viral DNA and spliced RNAs. Lectin-stimulated polymorphonuclear leukocytes from transgenic mice (SBL mouse) expressing a gp120-binding L3T4 mutant protein were infected with HIV-1 at the same frequency as lectin-stimulated human peripheral blood lymphocytes as determined by in situ PCR analyses. Supernatant p24gag and reverse transcriptase levels in HIV-infected mouse cell cultures, however, were routinely at background levels, unlike HIV-infected human cell cultures. Thus, gp120-binding mutant L3T4 proteins mediate viral entry in all mouse cells that were tested, but high-level viral replication is absent in these cells.

Degos disease is a lethal small vessel angiopathy targeting the skin, gastrointestinal tract, and central nervous system, potentially developing in the setting of known autoimmune disease, although forme fruste primary variants exist. Its pathogenetic basis is unknown. Four cases of Degos disease were encountered in archival material, representing 2 men, ages 38 and 43 years, and 2 females, ages 48 and 2 years; 3 patients died of disease. All had characteristic skin lesions with gastrointestinal involvement; other affected organs included brain in one and pericardium and pleura in another. Skin biopsies showed pauci-inflammatory thrombogenic microangiopathy with endothelial cell injury. Extracutaneous organs demonstrated fibromucinous occlusive arteriopathy. Prominent vascular C5b-9 was seen in the skin, gastrointestinal tract, and brain. All cases had evidence of high expression of interferon-α (based on tissue expression of MXA, a type I interferon-inducible protein), endothelial tubuloreticular inclusions, and an interferon gene signature in peripheral blood mononuclear cells. The MXA expression paralleled the pattern of C5b-9 deposition. Degos disease is a distinct vascular injury syndrome whereby a dysregulated interferon-α response in concert with membranolytic attack complex deposition may contribute to the unique vascular changes. Understanding the pathophysiology of the disease process could lead to more directed therapies, including terminal complement inhibition with agents such as eculizumab.

Reovirus is a systemically delivered oncolytic agent with evidence of activity in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, as well as activating anti-tumor innate and adaptive immunity. Having previously shown that intravenously delivered reovirus selectively accesses colorectal cancer metastatic to the liver in patients, in this study we explored the potential of systemic reovirus for the treatment of both primary and secondary brain tumors. We first showed that intravenous reovirus can be detected in tumors implanted into the brains of immunocompetent mice. We then tested our current most potent reovirus-based therapy (systemic reovirus plus granulocyte-macrophage colony stimulating factor [GMCSF] in reovirus pre-immune mice), and showed effective therapy in immunocompetent murine models of primary glioma, and of both directly reovirus sensitive, and insensitive, models of melanoma brain metastases. We also found that addition of reovirus/GMCSF to clinical ‘standard of care’ (radiotherapy and temozolamide chemotherapy) significantly enhances survival. In parallel to these pre-clinical experiments, we have initiated an open-label, non-randomized study of intravenous reovirus administered to patients prior to planned surgery for recurrent high grade glioma or metastatic brain tumors, to test whether the data showing access of the agent to tumors in the brains in mice, also applies to humans. In total, 10 patients will be treated with a single infusion of 1x1010 TCID50 of reovirus, of which 9 have completed the study to date. The primary objective is the presence of reovirus in the resected tumors as assessed by immunohistochemistry, RNA in-situ hybridization and retrieval of infectious virions. Early analysis of the first 3 patients, comprising one glioblastoma multiforme, one grade 3 oligodendroglioma and one colorectal brain metastasis, has shown that all 3 resected tumors contained reovirus RNA and protein. There was also evidence for productive reovirus infection in 2 of the tumors. Within all patients to date, the only grade 3-4 adverse reaction has been neutropaenia in 1 patient. Further tissue analysis is ongoing, as is testing of blood samples from these patients to further charactize how reovirus is carried in the blood and protected from neutralizing antibodies. This clinical study shows, for the first time that an oncolytic virus, reovirus, infects and replicates in brain tumors following intravenous administration. Together with pre-clinical data showing the efficacy of systemic reovirus in combination with GMCSF/radiation/temozolamide, these findings support the future development of trials and combination studies using reovirus in patients with high grade gliomas and brain metastases. Citation Format: Adel Jebar, Liz Ilett, Tim Kottke, Emma West, Karen Scott, Simon Thomson, Matt Coffey, Gerard Nuovo, Susan Short, Richard Vile, Alan Melcher. Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A49.

Background: MicroRNAs (miRs) are small non coding RNAs involved in cell homeostasis. miRs are deregulated in colorectal cancer (CRC). Our study aimed at identifying miRs with a driver role in carcinogenesis altered by similar mechanisms in both human and mouse CRC. Goal of the study was to use CRC mouse models for the pre-clinical development of anti-miRs as therapeutic drugs. Methods: Azoximetane (AOM)/Dextran-Sulfate (DSS) treated mice or CDX2Cre-APC f/wt mice were used to study inflammation-associated and sporadic APC-related CRC. Human Inflammatory Bowel Disease associated (n=30), and sporadic (n=90) CRC with their matched normal tissues were collected according to Good Clinical Practice recommendation and subjected to RNA extraction using Trizol. miR and gene expression profiling was assessed by nCounter technology (Nanostring Seattle). AntimiR-135b and scrambled probes for in vivo studies were synthesized by Girindus. Results: miRs profiling from AOM/DSS and CDX2Cre-APC f/wt CRC. revealed that miR-135b is one of the most up-regulated miRs in both models. In humans miR-135b over-expression was found in both IBD and sporadic CRC and was associated with reduced Progression Free Survival and Overall Survival in CRC patients. Molecular studies in Mouse Embryo Fibroblast and human CRC cell lines highlighted the role of two major pathways in the upstream activation of miR-135b: APC-β-Catenin and SRC-PI3K. MiR-135b up-regulation resulted in reduced apoptosis and increased invasion and metastasis due to the down-regulation of TGFRB2, DAPK1, APC and HIF1AN. Silencing of miR-135b in vivo reduced tumor multiplicity and tumor load in the AOM/DSS CRC model. Mice treated with anti-miR-135b showed well differentiated tumors and acinar pattern while tumors in the control groups showed low differentiation and adenomatous pattern. Conclusions: Our data suggest that miR-135b is a key molecule whose activation is downstream of oncogenes and oncosuppressor genes frequently altered in CRC. Our study defines specific pathways that converge on the activation of the same microrna. The “in vivo” silencing of miR-135 shows preclinical efficacy with low toxicity and represents the first in vivo study for the use of antimiRs in CRC treatment Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Nicola Valeri, Roberta Gafa', Gerard Nuovo, Giovanni Lanza, Wendy Frankel, Peter K. Vogt, Joanna Groden, Michael Karin, Carlo M. Croce, Chiara Braconi, Pierluigi Gasparini, Sergei Grivennikov, Jonathan R. Hart, Alessio Paone, Francesca Lovat, Muller Fabbri. Anti-miR-135b in colon cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B14.

To the Editor . —In the article by Kirgan et al 1 in the July 1990 issue of theArchives, the authors note that human papillomavirus (HPV) antigen and DNA were present in a large percentage of colon carcinomas, normal colonic mucosa, and benign colon tumors. These findings are the opposite of what has been described by others in the genital tract and colon. Human papilloma-virus DNA and HPV antigens are rarely found in normal genital tract tissues using in situ hybridization or immunohistochemistry. 2,3 Further, when the antigen is detected, the DNA is invariably also detected. Using more sensitive techniques (Southern blot analysis and the polymerase chain reaction, which can detect as few as 10 viral particles per million cells compared with 10 viruses per cell with the in situ method), several investigators have not been able to detect HPV DNA in colon tumors. 4,5 When the results from these