[Liver transplantation. Current aspects of pretransplantation diagnosis and rejection]. / Lebertransplantation. Aktuelle Aspekte der Prätransplantationsdiagnostik und Abstoßung.

Liver transplantation is an established treatment option for patients with end-stage liver disease. The therapy management of these patients is interdisciplinary and requires pathologists to have both clinical and immunological knowledge. Continuous advances in treatment and increasing clinical experience are accompanied by the further development of pathological transplant diagnostics. This article presents and discusses the latest classification of T­cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), and aspects of pretransplant diagnostics.

Transbronchial cryobiopsy in fibrosing interstitial lung disease: modifications of the procedure lead to risk reduction.

Sixty-one subjects with fibrosing interstitial lung disease were prospectively analysed to determine the efficacy of transbronchial cryobiopsy (CryoTBB) and the effect of procedural modifications which were introduced after an interim analysis of the first 19 subjects. The modifications significantly reduced complication rates from 84% to 14% (p<0.001). 30-day-mortality was 2%. The algorithm with initial CryoTBB and surgical lung biopsy (SLB) as optional step-up procedure was feasible. CryoTBB led to a confident diagnosis in 46/61 subjects (75%). Only 21% out of all subjects were forwarded for SLB. As the modified CryoTBB reduced but not eliminated the risk of severe complications, tissue sampling should be limited to patients where confident diagnosis enables life prolonging therapy. Trial registration number: NCT01714518.

Comprehensive Biomarker Analyses in Patients with Advanced or Metastatic Non-Small Cell Lung Cancer Prospectively Treated with the Polo-Like Kinase 1 Inhibitor BI2536.

BACKGROUND: Polo like kinase 1 (PLK1) is frequently upregulated in tumors and is thus viewed as a promising therapeutic target in various cancers. Several PLK1 inhibitors have recently been developed and clinically tested in solid cancers, albeit with limited success. So far, no predictive biomarkers for PLK1 inhibitors have been established. To this end, we conducted a post-hoc biomarker analysis of tumor samples from non-small cell lung cancer (NSCLC) patients treated with the PLK1 inhibitor BI2536 in a phase II study. METHODS: We analyzed formalin-fixed paraffin-embedded surplus tumor tissue from 47 study patients using immunohistochemistry (IHC) and DNA sequencing of KRAS, EGFR, BRAF, and PIK3CA. RESULTS: KRAS-mutated patients showed numerically prolonged progression-free survival, but statistical significance was not established. Interestingly, when pathways rather than single genes were analyzed, a positive correlation between IHC staining of activated ERK (p-ERK) and mutated KRAS was detected, whereas KRAS mutation status was found to be negatively correlated with activated AKT (p-AKT). CONCLUSION: With this hypothesis-generating study in BI2531-treated patients, we could not establish a correlation between KRAS mutations and relevant clinical endpoints. Future clinical trials with concomitant systematic biosampling and comprehensive molecular analyses are required to identify biomarkers predictive for response to PLK1 inhibitors.

The role of transbronchial cryobiopsy and surgical lung biopsy in the diagnostic algorithm of interstitial lung disease.

BACKGROUND AND AIMS: It is not yet known if transbronchial cryobiopsy (TCB) is a reliable and safe diagnostic tool in the investigation of interstitial lung disease (ILD). To date, there have been no studies directly comparing the value of TCB with that of surgical lung biopsy (SLB). The study was initiated to determine whether the samples taken by TCB lead to a reliable diagnosis and whether SLB can be avoided in a relevant percentage of cases. METHODS: We analyzed 32 subjects with suspected ILD who underwent a TCB. Subjects' baseline characteristics, pathological findings after TCB and SLB, and complication rates were analyzed. The pathological inter-rater agreement was quantified statistically. RESULTS: The overall inter-rater agreement concerning TCB sample evaluation was good with a kappa value of 0.80. In 23/32 cases (72%), the findings from the TCB showed a strong congruence with all other clinical data, thereby enabling a definitive diagnosis. Eight of the remaining nine subjects gave their consent for an SLB, which led to a definitive histological diagnosis in six cases (75%). Following TCB, pneumothorax occurred in 6/32 subjects (19%) and endobronchial bleeding was moderate in 8/32 (25%) and was severe in 17/32 cases (53%). CONCLUSION: This is the first study to correlate histological results and complications following TCB and SLB in ILD subjects, some of whom underwent both procedures. TCB is a suitable diagnostic tool in ILD, potentially completely dispensing with the need for an SLB in some cases. In all cases, an interdisciplinary case evaluation is necessary as a final step.

A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors.

Hepatocellular carcinoma (HCC) is a major lethal cancer worldwide. Despite sophisticated diagnostic algorithms, the differential diagnosis of small liver nodules still is difficult. While imaging techniques have advanced, adjuvant protein-biomarkers as glypican3 (GPC3), glutamine-synthetase (GS) and heat-shock protein 70 (HSP70) have enhanced diagnostic accuracy. The aim was to further detect useful protein-biomarkers of HCC with a structured systematic approach using differential proteome techniques, bring the results to practical application and compare the diagnostic accuracy of the candidates with the established biomarkers. After label-free and gel-based proteomics (n=18 HCC/corresponding non-tumorous liver tissue (NTLT)) biomarker candidates were tested for diagnostic accuracy in immunohistochemical analyses (n=14 HCC/NTLT). Suitable candidates were further tested for consistency in comparison to known protein-biomarkers in HCC (n=78), hepatocellular adenoma (n=25; HCA), focal nodular hyperplasia (n=28; FNH) and cirrhosis (n=28). Of all protein-biomarkers, 14-3-3Sigma (14-3-3S) exhibited the most pronounced up-regulation (58.8×) in proteomics and superior diagnostic accuracy (73.0%) in the differentiation of HCC from non-tumorous hepatocytes also compared to established biomarkers as GPC3 (64.7%) and GS (45.4%). 14-3-3S was part of the best diagnostic three-biomarker panel (GPC3, HSP70, 14-3-3S) for the differentiation of HCC and HCA which is of most important significance. Exclusion of GS and inclusion of 14-3-3S in the panel (>1 marker positive) resulted in a profound increase in specificity (+44.0%) and accuracy (+11.0%) while sensitivity remained stable (96.0%). 14-3-3S is an interesting protein biomarker with the potential to further improve the accuracy of differential diagnostic process of hepatocellular tumors. This article is part of a Special Issue entitled: Medical Proteomics.

(Cleaved) CK18 serum and tissue expression levels differentiate acute HCV reinfection from acute rejection in liver allografts.

BACKGROUND & AIMS: Orthotopic liver transplantation (OLT) is the sole therapeutic option to cure end-stage liver diseases including HCV-related cirrhosis. Timely and precise differentiation of relevant acute HCV reinfection from acute rejection after OLT is vital for appropriate therapy. Aim of this study was to evaluate the usefulness of (non-) invasive apoptosis (M30) and necrosis (M65) determination in the differential diagnosis of acute (and chronic) HCV reinfection vs. acute rejection in liver allografts. METHODS: Serum samples and liver biopsy tissues were available from 76 patients including a control group (19× NAFL, 19× NASH, 16× acute rejection, 11× acute and 11× chronic HCV reinfection) and were analysed using M30- and M65 ELISAs (M30S, M65S) and M30-immunohistochemistry (M30H). Clinical and serological data were collected. RESULTS: M30S, M65S and M30H were highly correlated with diagnostic groups in the total cohort (all P < 0.0001). M30S, M65S and M30H were independently able to differentiate acute HCV reinfection from acute rejection (P = 0.048, P = 0.001, P = 0.010) with moderate to excellent diagnostic accuracy (sensitivity, specificity, cut-off-value in M30S: 70%, 75%, 1025 U/L; M65S: 100%, 92%, 1308 U/L; M30H: 73%, 88%, 0.3%). CONCLUSIONS: M30-, M65-ELISAs and M30-immunohistochemistry are potential useful tools in differentiating acute HCV reinfection from acute rejection facilitating both speed and accuracy of the diagnostic process for the clinician and hepatopathologist. In this context, M65S provided superior diagnostic characteristics compared to M30-based methods. However, being the first analysis of (cleaved) CK18 serum and tissue expression levels in this context, the results need to be verified in further studies.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

BACKGROUND AND METHODS: Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. RESULTS: A total of 250 µg DNA (concentration 5 µg/µL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. CONCLUSION: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Subnormothermic machine perfusion for preservation of porcine kidneys in a donation after circulatory death model.

Machine perfusion for preservation led to compelling success for the outcome of renal transplantation. Further refinements of methods to decrease preservation injury remain an issue of high interest. This study investigates functional and morphological aspects of kidneys preserved by subnormothermic (20 °C) machine perfusion (SNTM) compared with oxygenated hypothermic machine perfusion (HMPox) and cold storage (CS) in a donation after circulatory death (DCD) model. After 30 min of warm ischaemia, porcine kidneys were randomly assigned to preservation for 7 h by CS, HMPox or SNTM. Afterwards, kidneys were reperfused for 2 h with autologous blood in vitro for assessment of function and integrity. Application of SNTM for preservation led to significantly higher blood flow and urine output compared with both other groups. SNTM led to a twofold increased creatinine clearance compared with HMPox and 10-fold increased creatinine clearance compared with CS. Structural integrity was best preserved by SNTM. In conclusion, this is the first study on SNTM for kidneys from DCD donors. SNTM seems to be a promising preservation method with the potential to improve functional parameters of kidneys during reperfusion.

Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. METHODS: We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. RESULTS: Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. CONCLUSIONS: The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.

A new preservation solution for lung transplantation: evaluation in a porcine transplantation model.

BACKGROUND: Lung preservation injury is still a major problem in lung transplantation. The aim of the current study was to evaluate the effects of a new preservation solution (Custodiol-N) for lung preservation. METHODS: Using an in vivo pig model, 7 lungs each were preserved for 24 hours after perfusion with: low-potassium dextran (LPD) solution as control (Group I); base solution of Custodiol-N without iron chelators (Group II); Custodiol-N (Group III); or Custodiol-N supplemented with dextran 40 (Group IV). Four animals received a sham operation. After left lung transplantation and contralateral lung exclusion, hemodynamics and blood gases were monitored for 6 hours; tissue samples were taken at the end of the experiments. RESULTS: All animals survived the transplantation procedure. Base solution- and Custodiol-N-preserved lungs (Groups II and III) showed graft function similar to that of LPD-preserved lungs (Group I), showing a trend toward improved values. Custodiol-N with dextran (Group IV) led to a significant reduction of mean pulmonary arterial pressure (20 ± 2 vs 28 ± 3 mm Hg, p < 0.01) and pulmonary vascular resistance (410 ± 51 vs 588 ± 83 dyne/s/cm(5), p < 0.01), and oxygenation ratio was significantly higher (536 ± 52 vs 313 ± 107 mm Hg at 6 hours, p < 0.01) and PCO(2) values were significantly lower (51 ± 9 vs 77 ± 5 mm Hg at 6 hours, p < 0.01) at 6 hours compared with LPD (Group I). Custodiol-N (Groups II to IV) showed a trend toward a lower wet/dry ratio and reduced oxidative stress; in the presence of dextran (Group IV), the difference was again statistically significant, when compared with LPD (Group I). CONCLUSIONS: Custodiol-N solution is a new alternative preservation solution for lung transplantation that offers significantly superior protection compared with LPD when dextran 40 is added.

Erythropoietin treatment improves liver regeneration and survival in rat models of extended liver resection and living donor liver transplantation.

BACKGROUND: Inadequate liver regeneration is still an unsolved problem in major liver resection and living donor liver transplantation (LDLT). Therefore, we have investigated the use of erythropoietin (EPO) as an exogenous stimulator of liver regeneration in rat models of liver resection and LDLT. METHODS: Rats were treated with EPO or heat-inactivated EPO-vehicles. Animals underwent 70% or 90% partial hepatectomy (PH) or 30% partial liver transplantation (pLTx). Serum and liver samples were taken to investigate liver function, liver-to-body weight ratio (LBWR), hepatocyte-proliferation (Ki-67), apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-assay), proregenerative cytokines (interleukin [IL]-6/tumor necrosis factor-alpha), and angiogenesis. Gene expression was assessed by in-house cDNA array and quantitative real-time polymerase chain reaction. As clinical parameters, LBWR and overall survival were determined. RESULTS: Erythropoietin led to improved liver regeneration as shown by an increased LBWR/Ki-67 after PH and pLTx. Liver damage, indicated by the serum activity of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase was reduced after PH. After surgery EPO treatment induced modulation of c-jun, IL-6, p53, and the antiapoptotic gene Bcl-XL, which was accompanied by a decreased apoptosis rate (0.56% vs. 1.03%; P<0.04). IL-6 production was increased at 12 hr, although no effects could be found concerning tumor necrosis factor-alpha production and angiogenesis. In addition, EPO-treated rats showed a significantly improved 28-day survival after 90% PH (92% vs. 67%) and pLTx (88% vs. 38%). CONCLUSIONS: Erythropoietin treatment significantly improved liver regeneration and survival after PH and pLTx and may therefore represent a promising strategy to optimize the clinical outcome after extended liver resection and LDLT in the future.