Detection of donor-derived cell-free DNA in the setting of multiple kidney transplantations.

Background: The routine use of donor-derived cell-free DNA (dd-cfDNA) assays to monitor graft damage in patients after kidney transplantation is being implemented in many transplant centers worldwide. The interpretation of the results can be complicated in the setting of multiple sequential kidney transplantations where accurate donor assignment of the detected dd-cfDNA can be methodologically challenging. Methods: We investigated the ability of a new next-generation sequencing (NGS)-based dd-cfDNA assay to accurately identify the source of the detected dd-cfDNA in artificially generated samples as well as clinical samples from 31 patients who had undergone two sequential kidney transplantations. Results: The assay showed a high accuracy in quantifying and correctly assigning dd-cfDNA in our artificially generated chimeric sample experiments over a clinically meaningful quantitative range. In our clinical samples, we were able to detect dd-cfDNA from the first transplanted (nonfunctioning) graft in 20% of the analyzed patients. The amount of dd-cfDNA detected from the first graft was consistently in the range of 0.1%-0.6% and showed a fluctuation over time in patients where we analyzed sequential samples. Conclusion: This is the first report on the use of a dd-cfDNA assay to detect dd-cfDNA from multiple kidney transplants. Our data show that a clinically relevant fraction of the transplanted patients have detectable dd-cfDNA from the first donor graft and that the amount of detected dd-cfDNA is in a range where it could influence clinical decision-making.

Assessment of treatment response in cardiac sarcoidosis based on myocardial 18F-FDG uptake.

Objective: Immunosuppressive therapy for cardiac sarcoidosis (CS) still largely consists of corticosteroid monotherapy. However, high relapse rates after tapering and insufficient efficacy are significant problems. The objective of this study was to investigate the efficacy and safety of non-biological and biological disease-modifying anti-rheumatic drugs (nb/bDMARDs) considering control of myocardial inflammation assessed by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) of the heart. Methods: We conducted a retrospective analysis of treatment response to nb/bDMARDs of all CS patients seen in the sarcoidosis center of the University Hospital Zurich between January 2016 and December 2020. Results: We identified 50 patients with CS. Forty-five patients with at least one follow-up PET/CT scan were followed up for a mean of 20.5 ± 12.8 months. Most of the patients were treated with prednisone and concomitant nb/bDMARDs. At the first follow-up PET/CT scan after approximately 6.7 ± 3 months, only adalimumab showed a significant reduction in cardiac metabolic activity. Furthermore, comparing all serial follow-up PET/CT scans (143), tumor necrosis factor inhibitor (TNFi)-based therapies showed statistically significant better suppression of myocardial 18F-FDG uptake compared to other treatment regimens. On the last follow-up, most adalimumab-treated patients were inactive (n = 15, 48%) or remitting (n = 11, 35%), and only five patients (16%) were progressive. TNFi was safe even in patients with severely reduced left ventricular ejection fraction (LVEF), and a significant improvement in LVEF under TNFi treatment was observed. Conclusion: TNFi shows better control of myocardial inflammation compared to nbDMARDs and corticosteroid monotherapies in patients with CS. TNFi was efficient and safe even in patients with severely reduced LVEF.

Case report of long-term postural tachycardia syndrome in a patient after messenger RNA coronavirus disease-19 vaccination with mRNA-1273.

Background: Postural tachycardia syndrome (POTS) is characterized by orthostatic intolerance and heart rate increase in an upright position without orthostatic hypotension. It has been described after coronavirus disease-19 (COVID-19) as well as after COVID-19 vaccination. Case summary: A 54-year-old female patient presented with a 9-months history of severe orthostatic intolerance since COVID-19 vaccination with messenger RNA (mRNA)-1273 (Spikevax, Moderna). Except for diet-controlled coeliac disease, the patient was healthy, had no allergies, and did not take regular medication. Tilt table testing revealed a significant heart rate increase to 168 bpm without orthostatic hypotension accompanied by light-headedness, nausea, and syncope, findings consistent with POTS. Potential underlying causes including anaemia, thyroid dysfunction, adrenal insufficiency, pheochromocytoma, (auto)-immune disease, chronic inflammation as well as neurological causes were ruled out. Echocardiography and cardiac stress magnetic resonance imaging (MRI) did not detect structural or functional heart disease or myocardial ischaemia. Forty-eight-hour-electrocardiogram (ECG) showed no tachycardias other than sinus tachycardia. Finally, genomic analysis did not detect an inherited arrhythmia syndrome. Serologic analysis revealed adequate immune response to mRNA-1273 vaccination without signs of previous severe acute respiratory syndrome-coronavirus-2 infection. While ivabradine was not tolerated and metoprolol extended release only slightly improved symptoms, physical exercise reduced orthostatic intolerance moderately. At a 5-months follow-up, the patient remained dependant on assistance for activities of daily living. Discussion: The temporal association of POTS with the COVID-19 vaccination in a previously healthy patient and the lack of evidence of an alternative aetiology suggests COVID-19 vaccination is the potential cause of POTS in this patient. To our knowledge, this is the first case reporting severe, long-term, and treatment-refractory POTS following COVID-19 vaccination with mRNA1273.

Donation type and the effect of pre-transplant donor specific antibodies - Data from the Swiss Transplant Cohort Study.

Introduction: The type of donation may affect how susceptible a donor kidney is to injury from pre-existing alloimmunity. Many centers are, therefore, reluctant to perform donor specific antibody (DSA) positive transplantations in the setting of donation after circulatory death (DCD). There are, however, no large studies comparing the impact of pre-transplant DSA stratified on donation type in a cohort with a complete virtual cross-match and long-term follow-up of transplant outcome. Methods: We investigated the effect of pre-transplant DSA on the risk of rejection, graft loss, and the rate of eGFR decline in 1282 donation after brain death (DBD) transplants and compared it to 130 (DCD) and 803 living donor (LD) transplants. Results: There was a significant worse outcome associated with pre-transplant DSA in all of the studied donation types. DSA directed against Class II HLA antigens as well as a high cumulative mean fluorescent intensity (MFI) of the detected DSA showed the strongest association with worse transplant outcome. We could not detect a significant additive negative effect of DSA in DCD transplantations in our cohort. Conversely, DSA positive DCD transplants appeared to have a slightly better outcome, possibly in part due to the lower mean fluorescent intensity (MFI) of the pre-transplant DSA. Indeed when DCD transplants were compared to DBD transplants with similar MFI (<6.5k), graft survival was not significantly different. Discussion: Our results suggest that the negative impact of pre-transplant DSA on graft outcome could be similar between all donation types. This suggests that immunological risk assessment could be performed in a similar way regardless of the type of donor kidney transplantation.

The impact of pre-transplant donor specific antibodies on the outcome of kidney transplantation - Data from the Swiss transplant cohort study.

Background: Pre-transplant donor specific antibodies (DSA), directed at non-self human leukocyte antigen (HLA) protein variants present in the donor organ, have been associated with worse outcomes in kidney transplantation. The impact of the mean fluorescence intensity (MFI) and the target HLA antigen of the detected DSA has, however, not been conclusively studied in a large cohort with a complete virtual cross-match (vXM). Methods: We investigated the effect of pre-transplant DSA on the risk of antibody-mediated rejection (ABMR), graft loss, and the rate of eGFR decline in 411 DSA positive transplants and 1804 DSA negative controls. Results: Pre-transplant DSA were associated with a significantly increased risk of ABMR, graft loss, and accelerated eGFR decline. DSA directed at Class I and Class II HLA antigens were strongly associated with increased risk of ABMR, but only DSA directed at Class II associated with graft loss. DSA MFI markedly affected outcome, and Class II DSA were associated with ABMR already at 500-1000 MFI, whereas Class I DSA did not affect outcome at similar low MFI values. Furthermore, isolated DSA against HLA-DP carried comparable risks for ABMR, accelerated eGFR decline, and graft loss as DSA against HLA-DR. Conclusion: Our results have important implications for the construction and optimization of vXM algorithms used within organ allocation systems. Our data suggest that both the HLA antigen target of the detected DSA as well as the cumulative MFI should be considered and that different MFI cut-offs could be considered for Class I and Class II directed DSA.

A case report of severe vitamin D intoxication / Schwere Vitamin-D-Intoxikation : Ein Fallbericht.

A CASE REPORT OF SEVERE VITAMIN D INTOXICATION: A 58-year-old woman presented with a symptomatic, hypertensive crisis, acute kidney failure AKIN2 and a severe hypercalcemia. The parathyroid hormone levels were in the lower normal range with highly elevated Vitamin D levels. For more than half a year she was taking 100'000 IU Vitamin D daily. Under volume loading, calcium-low-diet, Denosumab for blocking calcium resorption from bone and Ketoconazol to inhibit activation of Vitamin D a normalization of the calcium levels as well as an improvement of renal function could be observed. Loss-of-function mutations in the genes CYP24A1 and SLC34A1, involved in vitamin D metabolism leading to hypercalcemia could not be found in this patient.

The role of epigenetics and immunological imbalance in the etiopathogenesis of psoriasis and psoriatic arthritis.

Psoriasis (Ps) and psoriatic arthritis (PsA) represent a clinical and immunopathogenic continuum, called psoriatic disease, cumulatively affecting approximately 3% of the general population. Psoriatic disease is a chronic inflammatory disorder affecting the skin and musculoskeletal system. The immuno-pathogenesis is characterized by an activation of the TNF/IL-23/IL-17 cytokine axis, leading to an immunologic imbalance of T-cells resident in all affected tissues, mainly entheses. In the majority of cases, skin Ps predates rheumatological manifestations. Secondary to the higher incidence and the availability of mouse models, there is stronger data available on skin Ps, and data are, in most cases, relevant also to PsA. In a widely accepted model, environmental trigger factors like infections or trauma are capable of initiating an inflammatory cascade, ultimately creating a sustained state of chronic inflammation in genetically susceptible individuals. Besides well-known genetic susceptibility loci, epigenetic DNA modifications, which are associated with Ps development have been characterized recently and will be discussed in this article. The current evidence is promising in the possibility to provide new therapeutic avenues and fill the unmet need of patients, for whom current treatments either do not allow the disease to be controlled or must be continued for life.

BRAF inhibition sensitizes melanoma cells to α-amanitin via decreased RNA polymerase II assembly.

Despite the great success of small molecule inhibitors in the treatment of patients with BRAFV600E mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAFV600E inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAFV600E oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAFV600E inhibitors.

Colorectal cancer cells display chaperone dependency for the unconventional prefoldin URI1.

Chaperone dependency of cancer cells is an emerging trait that relates to the need of transformed cells to cope with the various stresses associated with the malignant state. URI1 (unconventional prefoldin RPB5 interactor 1) encodes a member of the prefoldin (PFD) family of molecular chaperones that acts as part of a heterohexameric PFD complex, the URI1 complex (URI1C), to promote assembly of multiprotein complexes involved in cell signaling and transcription processes. Here, we report that human colorectal cancer (CRCs) cell lines demonstrate differential dependency on URI1 and on the URI1 partner PFD STAP1 for survival, suggesting that this differential vulnerability of CRC cells is directly linked to URI1C chaperone function. Interestingly, in URI1-dependent CRC cells, URI1 deficiency is associated with non-genotoxic p53 activation and p53-dependent apoptosis. URI1-independent CRC cells do not exhibit such effects even in the context of wildtype p53. Lastly, in tumor xenografts, the conditional depletion of URI1 in URI1-dependent CRC cells was, after tumor establishment, associated with severe inhibition of subsequent tumor growth and activation of p53 target genes. Thus, a subset of CRC cells has acquired a dependency on the URI1 chaperone system for survival, providing an example of 'non-oncogene addiction' and vulnerability for therapeutic targeting.

Expression of the Stem Cell Factor Nestin in Malignant Pleural Mesothelioma Is Associated with Poor Prognosis.

BACKGROUND: The epithelioid and sarcomatoid histologic variants of malignant pleural mesothelioma (MPM) can be considered as E- and M-parts of the epithelial-mesenchymal transition (EMT) axis; the biphasic being an intermediate. EMT is associated with an increase of stem cell (SC) traits. We correlated the neural crest SC marker nestin and the EMT marker periostin with histology, type of neo-adjuvant chemotherapy (CT) and overall survival (OS) of MPM patients. PATIENTS AND METHODS: Tumor tissues of a historic cohort 1 (320 patients) and an intended induction chemotherapy followed by extrapleural pneumonectomy (EPP) cohort 2 (145 patients) were immunohistochemically H-scored (intensity of immunoreactivity multiplied by frequency of stained cells). Paired chemo-naïve biopsies and -treated surgical specimens were available for 105/145 patients. CT included platinum/gemcitabine (Pla/Gem) or platinum/pemetrexed (Pla/Pem). RESULTS: Expression of any cytosolic nestin progressively increased from epithelioid to biphasic to sarcomatoid MPM in cohort 1, whereas the diagnostic markers calretinin and podoplanin decreased. In cohort 2, Pla/Pem CT increased the expression level of nestin in comparison to Pla/Gem, whereas the opposite was found for periostin. In Pla/Pem treated patients, nestin was higher in biphasic MPM compared to epithelioid. In addition to non-epithelioid histology, any expression of nestin in chemo-naïve biopsies (median overall survival: 22 vs. 17 months) and chemo-treated surgical specimens (18 vs. 12 months) as well as high periostin in biopsies (23 vs. 15 months) were associated with poor prognosis. In the multivariate survival analysis, any nestin expression in chemo-naïve biopsies proved to be an independent prognosticator against histology. In both pre- and post-CT situations, the combination of nestin or periostin expression with non-epithelioid histology was particularly/ dismal (all p-values <0.05). CONCLUSIONS: The SC marker nestin and the EMT marker periostin allow for further prognostic stratification among histologic variants of MPM. Their expression level is influenced by neo-adjuvant chemotherapy.

Importance of excision repair cross-complementation group 1 and ribonucleotide reductase M1 as prognostic biomarkers in malignant pleural mesothelioma treated with platinum-based induction chemotherapy followed by surgery.

OBJECTIVES: Survival and response to platinum-based induction chemotherapy are heterogeneous among patients with malignant pleural mesothelioma. The aim of the present study was to assess the prognostic role of DNA repair markers, such as excision repair cross-complementation group 1 and ribonucleotide reductase M1, in multimodally treated patients with malignant pleural mesothelioma. METHODS: Tumor tissue of a malignant pleural mesothelioma cohort (n = 107) treated with platinum/gemcitabine (n = 46) or platinum/pemetrexed (n = 61) induction chemotherapy followed by extrapleural pneumonectomy was assembled on a tissue microarray. Immunohistochemical expression of excision repair cross-complementation group 1 (nuclear) and ribonucleotide reductase M1 (nuclear and cytoplasmic) was assessed for its prognostic impact (association with overall survival or freedom from recurrence). RESULTS: Patients with high nuclear ribonucleotide reductase M1 expression before chemotherapy showed significantly longer freedom from recurrence (P = .03). When specifically analyzed in the subgroup of patients receiving platinum/gemcitabine followed by extrapleural pneumonectomy, high nuclear ribonucleotide reductase M1 was associated with prolonged freedom from recurrence (P = .03) and overall survival (P = .02). Low excision repair cross-complementation group 1 expression in prechemotherapy tumor tissues was associated with significantly longer freedom from recurrence (P = .04). Nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 were independent prognosticators of freedom from recurrence in addition to pT stage in multivariate analysis. CONCLUSIONS: In the present study, nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 expression were identified as independent prognosticators for freedom from recurrence of malignant pleural mesothelioma in patients undergoing induction chemotherapy followed by extrapleural pneumonectomy.

PI3K/mTOR signaling in mesothelioma patients treated with induction chemotherapy followed by extrapleural pneumonectomy.

INTRODUCTION: The prognostic significance of activity biomarkers within the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway was assessed in two independent cohorts of malignant pleural mesothelioma (MPM) patients uniformly treated with a multimodal approach. We specifically assessed expression signatures in a unique set of pre- and postchemotherapy tumor samples. METHODS: Biomarker expression was assessed in samples of two independent cohorts of 107 (cohort 1) and 46 (cohort 2) MPM cases uniformly treated with platinum-based induction chemotherapy followed by extrapleural pneumonectomy from two different institutions, assembled on tissue microarrays. Expression levels of phosphatase and tensin homologue (PTEN), phospho-mTOR, and p-S6 in addition to marker of proliferation (Ki-67) and apoptosis (cleaved caspase-3) were evaluated by immunohistochemistry and correlated with overall survival (OAS) and progression-free survival (PFS). To assess PTEN genomic status, fluorescence in situ hybridization was performed. RESULTS: Survival analysis showed that high p-S6 and Ki-67 expression in samples of treatment naïve patients of cohort 1 was associated with shorter PFS (p = 0.02 and p = 0.04, respectively). High Ki-67 expression after chemotherapy remained associated with shorter PFS (p = 0.03) and OAS (p = 0.02). Paired comparison of marker expression in samples before and after induction chemotherapy of cohort 1 revealed that decreased cytoplasmic PTEN and increased phospho-mTOR expression was associated with a worse OAS (p = 0.04 and p = 0.03, respectively). CONCLUSIONS: These novel data reveal a prognostic significance of expression changes of PI3K/mTOR pathway components during induction chemotherapy if confirmed in other patient cohorts and support the growing evidence to target the PI3K/mTOR pathway in the treatment of MPM.