Sebastian Winkler

Combined genome and transcriptome analysis of single disseminated cancer cells from bone marrow of prostate cancer patients reveals unexpected transcriptomes

Bone is the most frequent site of metastasis in prostate cancer and patients with bone metastases are deemed incurable. Targeting prostate cancer cells that disseminated to the bone marrow before surgery and before metastatic outgrowth may therefore prevent lethal metastasis. This prompted us to directly analyze the transcriptome of disseminated cancer cells (DCC) isolated from patients with nonmetastatic (UICC stage M0) prostate cancer. We screened 105 bone marrow samples of patients with M0-stage prostate cancer and 18 bone marrow samples of patients without malignancy for the presence of EpCAM(+) single cells. In total, we isolated 270 cells from both groups by micromanipulation and globally amplified their mRNA. We used targeted transcriptional profiling to unambiguously identify DCCs for subsequent in-depth analysis. Transcriptomes of all cells were examined for the expression of EPCAM, KRT8, KRT18, KRT19, KRT14, KRT6a, KRT5, KLK3 (PSA), MAGEA2, MAGEA4, PTPRC (CD45), CD33, CD34, CD19, GYPC, SCL4A1 (band 3), and HBA2. Using these transcripts, we found it impossible to reliably identify true DCCs. We then applied combined genome and transcriptome analysis of single cells and found that EpCAM(+) cells from controls expressed transcripts thought to be epithelial-specific, whereas true DCCs may express hematopoietic transcripts. These results point to an unexpected transcriptome plasticity of epithelial cancer cells in bone marrow and question common transcriptional criteria to identify DCCs.

The impact of hypoxia on mesenchymal progenitor cells of human skeletal tissue in the pathogenesis of heterotopic ossification

PurposeMesenchymal progenitor cells (MPCs) are capable of differentiating into osteo/chondrogenic cells to contribute substantially to heterotopic ossification (HO). This study aimed to examine the impact of hypoxia on MPCs in the aetiology of HO.MethodsMPCs from human normal and HO skeletal tissue were cultivated under normoxia and hypoxia. Gene expression of factors which have a key role in HO aetiology (BMPs, COX-1 and COX-2, etc.) were examined by real-time PCR. Tissue of both groups was analysed by immunohistochemistry.ResultsUnder hypoxia, COX-1, -2 and SOX-9 gene expression was elevated in HO MPCs, whereas in normal muscle tissue only COX-2 was upregulated. MPCs from HO had a significantly elevated gene expression of BMP-4 and decreased expression of BMP-1 and HIF-1 under hypoxia compared to normal MPCs. Immunohistochemistry detected no significant differences between normal and HO tissue.ConclusionsHypoxia causes an enhanced gene expression of factors, which have a key role in HO pathophysiology. A better understanding of this entity will possibly allow reducing HO rates in orthopaedic and trauma surgery.

The impact of standard combined anteversion definitions on gait and clinical outcome within one year after total hip arthroplasty

PurposeDifferent target areas within the concept of combined cup and stem anteversion have been published for total hip arthroplasty (THA). We asked whether component positioning according to eight standard combined anteversion rules is associated with (1) more physiological gait patterns, (2) higher improvement of gait variables and (3) better clinical outcome after THA.MethodsIn a prospective clinical study, 60 patients received cementless THA through an anterolateral MIS approach in a lateral decubitus position. Six weeks postoperatively, implant position was analysed using 3D-CT by an independent external institute. Preoperatively, six and 12 months postoperatively range of motion, normalized walking speed and hip flexion symmetry index were measured using 3D motion-capture gait analysis. Patient-related outcome measures (HHS, HOOS, EQ-5D) were obtained by an observer blinded to 3D-CT results. Eight combined anteversion definitions and Lewinnek’s “safe zone” were evaluated regarding their impact on gait patterns and clinical outcome.ResultsCombined cup and stem anteversion according to standard combined anteversion definitions as well as cup placement within Lewinnek’s “safe zone” did not influence range of motion, normalized walking speed and/or hip flexion symmetry index six and 12 months after THA. Similarly, increase of gait parameters within the first year after THA was comparable between all eight combined anteversion rules. Clinical outcome measures like HHS, HOOS and EQ-5D did not show any benefit for either of the combined anteversion definitions.ConclusionsStandard combined cup and stem anteversion rules do not improve postoperative outcome as measured by gait analysis and clinical scores within one year after THA.

Perioperative blood loss and gastrointestinal tolerability of etoricoxib and diclofenac in total hip arthroplasty (ETO-DIC study): a single-center, prospective double-blinded randomized controlled trial

Abstract Objective: Non-selective NSAIDs can cause serious gastrointestinal side-effects. Selective COX-2 blockers are a reasonable alternative for pain treatment. They do not seem to affect platelet function and consequently cause a lower perioperative blood loss than non-selective NSAIDs. This study compared etoricoxib and diclofenac during a perioperative (9 days) period after THA to investigate total blood loss and gastrointestinal tolerability. The hypothesis was that etoricoxib is superior to diclofenac. Methods: A total of 100 patients (50 in each group) were included in this trial. Etoricoxib (90 mg) was administered once and diclofenac sodium (75 mg) twice daily for 9 days. Total blood loss during and after primary cementless THA was detected. The rate of adverse events (AEs) and serious adverse events (SAEs) was analyzed to detect gastrointestinal tolerability. Results: The mean total blood loss (calculated) was 1548 ± SD 468 ml in the etoricoxib (ETO) group and 1649 (SD 547) ml in the diclofenac (DIC) group. The mean duration of THA was 81 min (SD 29) in the DIC and 75 min (SD 30) in the ETO group. Hence, the mean calculated total blood loss was 101 ml higher in the DIC group. This difference was not statistically significant (p = 0.334). Fifty-six patients (28 in each group) received a cell saver retransfusion, but only one patient (ETO group) needed an additional red blood cell transfusion. The hidden blood loss was 1067 ml (SD 603) in the DIC group and 999 ml (SD 378) in the ETO group. The gastrointestinal tolerability (number of adverse and serious adverse events) was not significantly different between groups. Conclusion: There was no statistically significant difference in perioperative blood loss after primary THA under etoricoxib (90 mg) compared to diclofenac (75 mg). Furthermore, no gastrointestinal superiority of etoricoxib could be detected during a short period of 9 days.

Irradiation in the treatment of arthrofibrosis after total knee arthroplasty: a preliminary trial

Postoperative arthrofibrosis after primary and revision total knee arthroplasty is a severe complication for the affected patient. Several risk factors have been identified for this diagnosis. We present a series of 3 patients with arthrofibrosis after (revision) total knee arthroplasty. Approximately 2 hours before the operative revision procedure, all patients were irradiated once with 7.01 gray. At latest follow-up, all 3 patients showed a distinct improvement of range of motion. Before revision total knee arthroplasty due to arthrofibrosis, a single dose of pre- or postoperative irradiation should be considered.