Loss of TRAIL-Receptors Is a Recurrent Feature in Pancreatic Cancer and Determines the Prognosis of Patients with No Nodal Metastasis after Surgery

Introduction

Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.

Aims and Methods

Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.

Results

The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.

Conclusion

This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

Identification of four novel splice site mutations in the ornithine transcarbamylase gene

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows X-linked inheritance with frequent new mutations. Using polymerase chain reaction (PCR) amplification of the individual exons including adjacent intron sequences followed by direct sequencing of the amplimers we identified four new mutations affecting donor splice sites of introns 2, 5, 6, and 8. The mutation at the first position of intron 2 was a G to A exchange associated with acute neonatal hyperammonemia in a male patient at the age of 5 months. A G to C substitution in intron 5 was detected in a boy who developed 2 days after birth hypotonia, and respiratory distress, followed by severe hyperammonemia and terminal coma. The intron 6 mutation, a G to T substitution, was detected in a girl presenting with first episodes of vomiting and agitation at the age of 2 months. The mutation in intron 8, also a G to T transition, caused fatal hyperammonemia and early death at the age of 15 days in a male patient. We present four donor splice site mutations resulting in severe neonatal or very early onset of the disease in three boys and in one female patient. As the GT dinucleotide of the 5 donor splice site is invariant and required for correct splicing the described mutations may lead to improperly spliced mRNAs and aberrant gene products.     

Circulating Serum MicroRNA-130a as a Novel Putative Marker of Extramedullary Myeloma

Poor outcome of extramedullary disease in multiple myeloma patients and lack of outcome predictors prompt continued search for new markers of the disease. In this report, we show circulating microRNA distinguishing multiple myeloma patients with extramedullary disease from myeloma patients without such manifestation and from healthy donors. MicroRNA-130a was identified by TaqMan Low Density Arrays and verified by quantitative PCR on 144 serum samples (59 multiple myeloma, 55 myeloma with extramedullary disease, 30 healthy donors) in test and validation cohorts as being down-regulated in myeloma patients with extramedullary disease. Circulating microRNA-130a distinguished myeloma patients with extramedullary disease from healthy donors with specificity of 90.0% and sensitivity of 77.1%, patients with extramedullary disease from newly diagnosed multiple myeloma patients with specificity of 77.1% and sensitivity of 34.3% in the test cohort and with specificity of 91.7% and sensitivity of 30.0% in the validation cohort of patients. Circulating microRNA-130a in patients with extramedullary myeloma was associated with bone marrow plasma cells infiltration. Further, microRNA-130a was decreased in bone marrow plasma cells obtained from patients with extramedullary myeloma in comparison to bone marrow plasma cells of myeloma patients without such manifestation, but it was increased in tumor site plasma cells of patients with extramedullary disease compared to bone marrow plasma cells of such patients (p<0.0001). Together, our data suggest connection between lower level of microRNA-130a and extramedullary disease and prompt further work to evaluate this miRNA as a marker of extramedullary disease in multiple myeloma.     

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.     

Synergistic Cooperation between Two ClpB Isoforms in Aggregate Reactivation

Bacterial AAA⁺ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA⁺ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.     

Allogeneic T cells treated with amotosalen prevent lethal cytomegalovirus disease without producing graft-versus-host disease following bone marrow transplantation

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 x 10(6) pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-gamma production by MCMV-primed CD8(+) T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log(10) and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44(high)) CD8(+) T cells persisted to day +100 following infusion, and expressed IFN-gamma when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.