Hagen S. Bachmann

Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging

Optoacoustic imaging strategies can be used to identify metastasis in excised lymph nodes and to determine SLN status in patients noninvasively. Imaging melanoma metastasis Avoiding invasive biopsy altogether, an imaging technique that relies on endogenous biomolecules to generate acoustic signals could be used to detect metastases in the body. Stoffels et al. devised a multispectral optoacoustic tomography (MSOT) approach that could image the pigment melanin in lymph nodes. Melanin would only be present in the lymph nodes if the primary cancer—melanoma—had spread to distant locations. The authors used handheld MSOT detectors and a near-infrared fluorophore (which pools in lymph nodes) to image metastases in patients, and complemented these optoacoustic images with ultrasound to gain a complete picture of each lymph node’s status. Such a noninvasive approach could reduce the number of patients subjected to sentinel lymph node surgical excision by “ruling out” metastasis. Sentinel lymph node (SLN) excision is included in various cancer guidelines to identify microscopic metastatic disease. Although effective, SLN excision is an invasive procedure requiring radioactive tracing. Novel imaging approaches assessing SLN metastatic status could improve or replace conventional lymph node excision protocols. In our first-in-human study, we used noninvasive multispectral optoacoustic tomography (MSOT) to image SLNs ex vivo and in vivo in patients with melanoma, to determine metastatic status. MSOT significantly improved the tumor metastasis detection rate in excised SLN (506 SLNs from 214 melanoma patients) compared with the conventional EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group protocol (22.9% versus 14.2%). MSOT combined with the near-infrared fluorophore indocyanine green reliably visualized SLNs in vivo in 20 patients, up to 5-cm penetration and with 100% concordance with 99mTc-marked SLN lymphoscintigraphy. MSOT identified cancer-free SLNs in vivo and ex vivo without a single false negative (189 total lymph nodes), with 100% sensitivity and 48 to 62% specificity. Our findings indicate that a noninvasive, nonradioactive MSOT-based approach can identify and determine SLN status and confidently rule out the presence of metastasis. The study further demonstrates that optoacoustic imaging strategies can improve the identification of SLN metastasis as an alternative to current invasive SLN excision protocols.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in head and neck squamous cell carcinomas.

BackgroundChronic inflammation plays an important role in head and neck squamous cell carcinomas (HNSCC). This study addresses the impact of two single nucleotide polymorphisms (SNP) Asp299Gly and Thr399Ile of the toll-like receptor (TLR) 4 gene on the clinical outcome while accounting for the influence of adjuvant systemic therapy in a large cohort of HNSCC patients.MethodsGenotype analysis was done using DNA from tissue samples from 188 patients with HNSCC; TLR4 protein expression was assessed immunohistochemically in tissue microarrays. Classical survival models were used for statistical analyses.ResultsTen percent of patients with HNSCC presented with the TLR4 299Gly and 17% with the TLR4 399Ile allele. Patients with the heterozygous genotype TLR4 Asp299Gly had a significantly reduced disease-free and overall survival. Also, patients with the heterozygous genotype TLR4 Thr399Ile had a reduced disease-free survival. Notably, these associations seem to be attributable to relatively poor therapy response as e.g. reflected in a significantly shorter DFS among HNSCC patients carrying the Asp299Gly variant and receiving adjuvant systemic therapy.ConclusionAccording to this study, TLR4 299Gly und 399Ile alleles may serve as markers for prognosis of head and neck cancer in patients with adjuvant systemic therapy, particularly chemotherapy, and might indicate therapy resistance.

Regulatory BCL2 promoter polymorphism (-938C>A) is associated with adverse outcome in patients with prostate carcinoma.

Molecular markers predictive of prostate cancer prognosis are urgently needed. Overexpression of the antiapoptotic protein, Bcl‐2, has repeatedly been shown to be associated with adverse outcome in this malignancy. We hypothesized that a regulatory BCL2 −938C>A promoter polymorphism, which significantly affects promoter activity and Bcl‐2 expression in different malignancies, may influence survival. Reporter assays and electrophoretic mobility shift assays reveled that the −938C>A BCL2 promoter polymorphism significantly affects promoter activity and transcription factor binding in prostate cancer cells. Significantly higher BCL2 mRNA expression was observed in primary prostate carcinomas derived from patients with the AA, compared to CC, genotype. Survival analysis showed that the −938AA genotype was an independent, unfavorable prognostic factor for relapse‐free survival in a primary cohort of 142 patients and in an independent replication cohort of 148 patients, with hazard ratios (HR) of 4.4 (95% CI, 1.3–15.1; p = 0.018) and 4.6 (95% CI, 1.5–14.2; p = 0.009). Furthermore, the −938AA genotype was independently associated with worse overall survival in the replication series, with a HR of 10.9 (95% CI, 1.2–99.3; p = 0.034). We conclude that the BCL2 −938C>A polymorphism is an independent predictor of relapse‐free and overall survival in patients with prostate cancer. The BCL2 −938C>A polymorphism should be evaluated prospectively and may also have promise in assisting optimal patient choice for treatment with BCL2‐targeted drugs already in evaluation for prostate cancer treatment.

RANKL-associated suppression of particle-induced osteolysis in an aged model of Calcitonin and α-CGRP deficiency.

An aging population with higher bone turnover intensifies the need for joint replacement surgery. However, particle-induced osteolysis (PIO) remains a major cause of early implant loosening. Differences in bone remodeling between young and aged Calcitonin (CT)- and α-CGRP (Calcitonin gene-related peptide)-deficient mice (Calca(-/-)) might modify our previous findings regarding CT/α-CGRP in PIO. This may have important implications for PIO in an aging population. Four groups of twelve-month-old wild-type and Calca(-/-) mice underwent either SHAM surgery with and without CT, or polyethylene-particle implantation with related treatment. Morphometric changes were detected using μ-CT, histomorphometric analysis and by counting TRAP(+) cells (osteoclast-staining). Bone remodeling was assessed using serum and urinary markers. There was no osteolysis in aged particle-treated Calca(-/-) animals and the effect of CT on PIO was reduced compared to wild-type mice. However, there were significantly higher numbers of TRAP(+) cells in Calca(-/-) animals, and bone remodeling markers revealed a significant increase in OPG/OCN and a significant reduction in RANKL compared to aged wild-type mice. CT/α-CGRP modulates bone cell activity in PIO in aged mice in a way that is distinct from young animals. This may have implications for the treatment of PIO in the periprosthetic surface of joint replacements in an aging population.

The XbaI G>T Polymorphism of the Glucose Transporter 1 Gene Modulates 18F-FDG Uptake and Tumor Aggressiveness in Breast Cancer

We investigated the relevance of single-nucleotide polymorphisms (SNPs) in the glucose transporter 1 (GLUT1) gene to the uptake of 18F-FDG and tumor aggressiveness in breast cancer. Methods: In 52 individuals with breast cancer, a diagnostic PET/CT scan was obtained, and the standardized uptake value was determined as a measure of 18F-FDG uptake using a region-of-interest technique. Three GLUT1 SNPs (XbaI G>T, HpyCH4V A>T, and HaeIII T>C) were investigated in genomic DNA that was isolated from the paraffin-embedded specimens of all patients. Tumors were typed and graded according to the World Health Organization classifications. Results: The GG genotype of the XbaI G>T SNP was associated with increased tumor uptake of 18F-FDG, with a mean standardized uptake value of 11.7 (TT/GT genotypes, 5.9; P = 0.03). Furthermore, the GG genotype was positively related to enhanced tumor proliferation (mitotic count, P = 0.01). In line with this finding, the GG genotype was absent in grade 1 carcinomas and increasingly prevalent in tumors with higher malignancy (grade 2, 28.0%; grade 3, 50%; P = 0.04). Conclusion: This study found that the XbaI G>T SNP of the GLUT1 gene is associated with an increased 18F-FDG uptake and a more advanced tumor grade or growth in breast cancer. Thus, this genetic variant might favor aggressive phenotypes by modulating the efficiency of cancer cells to recruit glucose and escalate growth rate, suggesting the XbaI G>T SNP as a proliferation-related prognostic factor.

Methods to evaluate the pharmacology of oral antiplatelet drugs.

Principally, there are two reasons why the pharmacological response to antiplatelet drugs should be measured: on the one hand, an insufficient inhibition of platelet function may result in atherothrombotic complications; on the other hand, an excessive inhibition of platelet function may lead to bleeding complications. The clinical importance to measure the effects of antiplatelet drugs is demonstrated by increasingly growing evidence for an association of resistance to antiplatelet drugs with thromboembolic events. It is often claimed that there is no generally accepted definition of “resistance” and, instead, there is an ongoing semantic discussion about the correct term to be used to describe this phenomenon. From the pharmacological point of view, there is only one acceptable definition of “resistance” to antiplatelet drugs: the term “resistance” should be used when a drug is unable to hit its pharmacological target. Thus, laboratory methods used to evaluate the effects of antiplatelet drugs should be designed to measure the direct pharmacodynamic effect of a drug, rather than the consequences for global platelet function. Based on physiological/pathophysiological, pharmacological, and practical considerations, the authors propose the following assays to be used to measure the effects of oral antiplatelet drugs: for the detection of aspirin actions, thromboxane or arachidonic acid-induced responses (light aggregometry, whole-blood aggregometry) should be measured; for the detection of clopidogrel actions, VASP (vasodilator-stimulated phosphoprotein) phosphorylation (flow cytometry) or ADP-(adenosine diphosphate-)induced responses (light aggregometry, whole-blood aggregometry, possibly also flow cytometry) should be measured.ZusammenfassungEs gibt zwei prinzipielle Gründe für die Messung der pharmakologischen Wirksamkeit von Thrombozytenfunktionshemmern: Auf der einen Seite kann eine unzureichende Hemmung der Thrombozytenfunktion zu atherothrombotischen Komplikationen führen; auf der anderen Seite kann eine exzessive Hemmung der Thrombozytenfunktion in Blutungskomplikationen resultieren. Die klinische Bedeutung der Messung der Wirkungen von Thrombozytenfunktionshemmern zeigt sich in einer zunehmenden Evidenz für die Assoziation einer fehlenden pharmakologischen Wirksamkeit mit thromboembolischen Ereignissen. Es wird häufig postuliert, dass es keine allgemein akzeptierte Definition für „Resistenz““ gegenüber Thrombozytenfunktionshemmern gibt. Stattdessen wird eine semantische Diskussion über den korrekten Begriff zur Beschreibung des Phänomens geführt. Aus pharmakologischer Sicht gibt es jedoch eine klare Definition der Resistenz gegenüber Thrombozytenfunktionshemmern: Der Begriff „Resistenz“ sollte benutzt werden, wenn ein Pharmakon den für die klinische Wirksamkeit entscheidenden pharmakodynamischen Mechanismus nicht ausüben kann. Daher sollten bevorzugt solche Labormethoden eingesetzt werden, die möglichst direkt den pharmakodynamischen Effekt eines Pharmakons erfassen. Basierend auf physiologischen/pathophysiologischen, pharmakologischen und praktischen Überlegungen wird vorgeschlagen, dass zur Erfassung der Wirkungen von Acetylsalicylsäure die Thromboxanbildung oder die arachidonsäureinduzierte Thrombozytenaggregation (Lichtaggregometrie, Vollblutaggregometrie) bestimmt werden sollte. Zur Erfassung der Wirkungen von Clopidogrel erscheint die Bestimmung der VASP-(„vasodilator-stimulated phosphoprotein“-)Phosphorylierung (Durchflusszytometrie) oder die ADP-(Adenosindiphosphat-) induzierte Thrombozytenaggregation (Lichtaggregometrie, Vollblutaggregometrie) bzw. Thrombozytenaktivierung (Durchflusszytometrie) sinnvoll.

Calcitonin substitution in calcitonin deficiency reduces particle-induced osteolysis

BackgroundPeriprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice.MethodsWe used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells.ResultsBone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution.ConclusionsAs anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.

BCL2-938C > A and CALCA-1786T > C polymorphisms in aseptic loosened total hip arthroplasty

The search for influencing factors and new pathways in aseptic loosening of arthroplasties is a major focus of recent studies. Analyses of polymorphisms of genes revealed a correlation between a specific allele variant and aseptic loosening. The BCL2 gene encoding Bcl-2 with its BCL2 -938C > A polymorphism is a crucial factor of cell cycle control and cell survival. The CALCA -1786T > C polymorphism belongs to the CALCA gene encoding alpha-Calcitonin Gene Related Peptide (CGRP) and Calcitonin. Both proteins are important in bone metabolism and capable to influence the process of aseptic loosening. To date, no studies are reported for aseptic loosening with these two single nucleotide polymorphisms (SNPs). In a retrospective study we determined the distribution of the BCL2-938C > A and the CALCA-1786T > C polymorphisms in 87 subjects with aseptic loosened hip arthroplasties using RFLP and pyrosequencing analysis. Genotype distribution with prognosis of the hip arthroplasty showed neither an association with clinical characteristics of the patients nor the implantation technique. We were unable to detect any influence of these polymorphisms on time to aseptic loosening.

Gender-dependent association of the GNAS1 T393C polymorphism with early aseptic loosening after total hip arthroplasty.

The G‐protein Gαs is involved in the physiology and pathophysiology of bone. Especially, Gαs is a key regulator of interleukin‐6, which is a potent promoter of aseptic loosening. We hypothesized that the common single nucleotide polymorphism GNAS1 T393C could also affect time to aseptic loosening. Caucasian patients were genotyped for the GNAS1 T393C polymorphism. Time and median time to aseptic loosening were analyzed for dependency on GNAS1 genotypes. Time and median time were not significantly associated with genotypes. Additional analysis corrected for gender revealed, that the TT genotype was associated with significantly longer time (p = 0.048) as well as median time (p = 0.022) to aseptic loosening in female patients. In contrast to the findings in females, male TT genotype carriers had significantly shorter time (p = 0.018) and median time (p = 0.023) to aseptic loosening. Compared with TT genotype carriers heterozygous patients had a 6.25‐fold lower risk with a hazard ratio of 0.160 (p = 0.016) and male patients carrying the CC genotype had an 11‐fold lower risk with a hazard ratio of 0.088 (p = 0.006) in multivariate analysis. The present study suggests a significant gender‐dependent role of the T393C polymorphism in aseptic loosening. The apparently contradictory results in women and men and the finding that the GNAS1 T393C genotype is an independent factor for time to aseptic loosening in male patients assigned this polymorphism as an interesting target for further investigations in bone diseases.

Association of the CC genotype of the regulatory BCL2 promoter polymorphism (-938C>A) with better 2-year survival in patients with glioblastoma multiforme.

OBJECT Bcl-2 plays a key role in the downregulation of apoptosis and proliferation and leads to increased chemoresistance in glioblastoma multiforme (GBM). The authors investigated the role of a common regulatory single-nucleotide polymorphism (-938C>A), which is located in the inhibitory P2 promoter of BCL2. METHODS Data from 160 patients suffering from GBM were retrospectively evaluated. Study inclusion criteria consisted of available DNA and, in patients still alive, a follow-up of at least 24 months. Results were analyzed with respect to the basic clinical data, type of surgical intervention (gross-total resection [GTR] versus stereotactic biopsy [SB]), adjuvant therapy, MGMT promoter methylation, and survival at the 2-year follow-up. RESULTS At the 2-year follow-up, 127 (79.4%) of the 160 patients had died. Kaplan-Meier curves revealed a significantly higher rate of survival for homo- and heterozygous C-allele carriers (p = 0.031). In the GTR group, the survival rate was 47.1% for homozygous C-allele carriers, 32.0% for heterozygous C-allele carriers, and only 21.4% for homozygous A-allele carriers (p = 0.024). The SB group showed no genotype-dependent differences. Multivariable Cox regression revealed that the BCL2 (-938AA) genotype was an independent negative prognostic factor for 2-year survival in the GTR group according to the BCL2 (-938CC) genotype reference group (hazard ratio 2.50, 95% CI 1.14-5.48, p = 0.022). CONCLUSIONS These results suggested that the (-938C>A) polymorphism is a survival prognosticator as well as a marker for a high-risk group among patients with GBM who underwent GTR.