Stratifying esophago-gastric cancer treatment using a patient-derived organoid-based threshold.

BACKGROUND AND AIMS: This study sought to determine the value of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) for response prediction to neoadjuvant chemotherapy (neoCTx). METHODS: Endoscopic biopsies of patients with locally advanced EGC (n = 120) were taken into culture and PDOs expanded. PDOs' response towards the single substances of the FLOT regimen and the combination treatment were correlated to patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n = 13) and subsequently confirmed in an independent validation cohort (n = 13). RESULTS: EGC PDOs reflected patients' diverse responses to single chemotherapeutics and the combination regimen FLOT. In the exploratory cohort, PDOs response to single 5-FU and FLOT combination treatment correlated with the patients' pathological response (5-FU: Kendall's τ = 0.411, P = 0.001; FLOT: Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision in receiver operating characteristic (ROC) analysis was reached with an AUCROC of 0.994 (CI 0.980 to 1.000). The discriminative ability of PDO-based FLOT testing allowed the definition of a threshold, which classified in an independent validation cohort FLOT responders from non-responders with high sensitivity (90%), specificity (100%) and accuracy (92%). CONCLUSION: In vitro drug testing of EGC PDOs has a high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. Taking into account the high rate of successful PDO expansion from biopsies, the definition of a threshold that allows treatment stratification paves the way for an interventional trial exploring PDO-guided treatment of EGC patients.

Quality of life evaluation and lack of correlation with volumetric results after lymphovenous anastomoses in lymphedema therapy of the lower extremity.

OBJECTIVE: Chronic primary or secondary lymphedema has huge effects on patients' quality of life (QOL) because of the associated swelling and pain, decreased range of motion, and depression and anxiety and generally requires numerous adaptations. Many studies have shown a positive objective effect of lymphovenous anastomoses (LVAs) on chronic lymphedema. In the present study, we assessed the effect of LVAs on QOL in patients with primary or secondary lymphedema of the lower extremity at 6 months after surgery and examined the correlation between changes in the QOL and volumetric measurements. METHODS: Only patients with either primary or secondary lymphedema of the lower extremity who had undergone LVAs were included in the present study. To assess QOL, a specially designed questionnaire based on the Lymphedema Quality of Life Inventory was used to evaluate the subjective therapeutic results from the patients' perspective. Objective therapy success was assessed using three-dimensional volumetric measurements of the lower leg. The measuring points, for both the subjective and the objective measurements, were the day before and 6 months after therapy. RESULTS: The mean change in volume at 6 months after LVA was -6.5% ± 5.6% (P < .001). Significantly better QOL in terms of physical (37.6% ± 25.2%) and psychosocial (27.0% ± 43.0%) domains and practical restrictions (22.3% ± 24.8%) was found (P < .001 for all). No correlation was found between QOL improvement and volume decrease (P > .05). CONCLUSIONS: For patients with lymphedema of the lower extremity, LVAs can lead to a significant volumetric decrease and QOL improvement at 6 months after treatment with no demonstrable relationship between QOL improvement and volume reduction.

Efficacy of Immune Checkpoint Inhibitors Alone or in Combination With Chemotherapy in NSCLC Harboring ERBB2 Mutations.

INTRODUCTION: In contrast to other driver mutations, no targeted therapies have yet been approved in ERBB2-mutated NSCLC (HER2mu NSCLC). Nevertheless, several compounds have revealed promising early efficacy data, which need to be evaluated in the context of current standard approaches. Although data on the efficacy of immune checkpoint inhibitors (ICIs) in second or subsequent lines of treatment remain limited and conflicting, there are virtually no data on patient outcome under ICI/platinum-doublet combinations in the first-line setting. METHODS: We retrospectively evaluated outcomes of patients with HER2mu NSCLC treated with ICI alone or in combination with chemotherapy within the German National Network Genomic Medicine Lung Cancer consortium by means of overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: ICI either in combination with chemotherapy or as monotherapy was applied as first-line treatment in 27 patients, whereas 34 received single-agent ICI in second or subsequent lines. Patient characteristics were in line with previously published data. In treatment-naive patients receiving ICI in combination with chemotherapy, the ORR, median PFS, and OS rate at 1 year were 52%, 6 months, and 88%, respectively. In second or subsequent lines, ICI monotherapy was associated with an ORR of 16%, a median PFS of 4 months, and a median OS of 10 months. CONCLUSIONS: ICIs are effective as monotherapy and in combination with platinum-doublet chemotherapy. Therefore, ICI-based treatments may be found as the current standard of care and benchmark for targeted therapies in HER2mu NSCLC.

The clinical benefit of molecular re-assessments in management of progressive lung cancer.

Despite the enormous success of molecularly targeted therapy in advanced non-small cell lung cancer (NSCLC), long-term disease control remains challenging. Almost all patients on targeted therapy ultimately progress due to plethora of acquired resistance mechanisms. While acquired resistance mechanisms in BRAF-V600 mutant malignant melanomas treated with targeted therapy are well studied, little is known about resistance mechanisms in BRAF-V600 mutant lung cancer so far. Therefore, patients progressing on the standard BRAF and MEK inhibitor combination are uniformly switched to immune- and/or chemotherapy. We describe the case of a metastatic BRAF-V600E mutant pulmonary adenocarcinoma of the left lung with presumed progression of a single lung lesion at the right side during targeted therapy. Due to oligo-progression, resection was performed. Molecular re-assessment for analysis of acquired resistance mechanisms surprisingly revealed a genetically distinct second primary malignancy. Following curative resection of the right sided second primary NSCLC, primary tyrosine kinase inhibitor therapy was continued and to date the patient is still responding with a cumulative treatment duration of now 34 months. This case report illustrates that a thorough molecular re-assessment upon progression on targeted therapies may have a decisive influence on subsequent treatment decisions and should therefore be considered on a routine basis.

Routine Molecular Pathology Diagnostics in Precision Oncology.

BACKGROUND: Technical advances in the field of molecular genetics permit precise genomic characterization of malignant tumors. This has not only improved our understanding of tumor biology but also paved the way for molecularly stratified treatment strategies in routine clinical practice. METHODS: A selective search of PubMed to identify literature on molecular pathology methods, their indications, the challenges associated with molecular findings, and future developments. RESULTS: Tumors can be characterized with the aid of immunohistochemistry, in-situ hybridization, and sequencing of DNA or RNA. The benefits of molecularly stratified tumor treatment have been demonstrated by randomized clinical trials on numerous tumor entities, e.g., non-small-cell lung cancer, colorectal cancer, and breast cancer. Therefore, initiation of specific treatment for these entities should be preceded by molecular pathology biomarker analyses, generally carried out on tumor tissue. Randomized controlled trials and non-controlled studies show that enhanced progression-free survival ensues if the pharmacological treatment is oriented on the findings of molecular pathology diagnostics. In next-generation sequencing, numerous relevant gene sequences or even whole genes can be sequenced in parallel, dispensing with complex staged diagnostics and reducing the use of biomaterials. These new methods also complement the currently relevant predictive biomarkers by permitting the investigation of genetic alterations presently of interest in the context of clinical studies. Prior to widespread routine clinical application, however, sequencing of large gene panels or whole genomes or exomes need to be even more stringently validated. CONCLUSION: Quality-assured molecular pathology assays are universally available for the determination of currently relevant predictive biomarkers. However, the integration of extensive genomic analyses into routine molecular pathology diagnostics represents a future challenge in precision oncology.

Extracorporeal perfusion - reduced to a one-way infusion.

BACKGROUND: Extracorporeal perfusion (EP) is moving into focus of research in reconstructive and transplantation medicine for the preservation of amputates and free tissue transplants. The idea behind EP is the reduction of ischemia-related cell damage between separation from blood circulation and reanastomosis of the transplant. Most experimental approaches are based on a complex system that moves the perfusate in a circular course. OBJECTIVE AND METHODS: In this study, we aimed to evaluate if a simple perfusion by an infusion bag filled with an electrolyte solution can provide acceptable results in terms of flow stability, oxygen supply and viability conservation for EP of a muscle transplant. The results are compared to muscles perfused with a pump system as well as muscles stored under ischemic conditions after a one-time intravasal flushing with Jonosteril. RESULTS: With this simple method a sufficient oxygen supply could be achieved and functionality could be maintained between 3.35 times and 4.60 times longer compared to the control group. Annexin V positive nuclei, indicating apoptosis, increased by 9.7% in the perfused group compared to 24.4% in the control group. CONCLUSIONS: Overall, by decreasing the complexity of the system, EP by one-way infusion can become more feasible in clinical situations.

Tissue Viability of Free Flaps after Extracorporeal Perfusion Using a Modified Hydroxyethyl Starch Solution.

BACKGROUND: In free flap surgery, tissue is stored under hypothermic ischemia. Extracorporeal perfusion (EP) has the potential to extend storage time and the tissue's perspective of survival. In the present study, the aim is to improve a recently established, simplified extracorporeal perfusion system. METHODS: Porcine musculus rectus abdominis were stored under different conditions. One group was perfused continuously with a simplified one-way perfusion system for six hours, while the other received only a single flush but no further treatment. A modified hydroxyethyl starch solution was used as a perfusion and flushing solution. Vitality, functionality, and metabolic activity of both groups were analyzed. RESULTS: Perfused muscles, in contrast to the ischemically stored ones, showed no loss of vitality and significantly less functionality loss, confirming the superiority of storage under continuous perfusion over ischemic storage. Furthermore, in comparison to a previous study, the results were improved even further by using a modified hydroxyethyl starch solution. CONCLUSION: The use of EP has major benefits compared to the clinical standard static storage at room temperature. Continuous perfusion not only maintains the oxygen and nutrient supply but also removes toxic metabolites formed due to inadequate storage conditions.

MiR-130a in the adipogenesis of human SGBS preadipocytes and its susceptibility to androgen regulation.

Objectives: Adipogenesis is the differentiation process generating mature adipocytes from undifferentiated mesenchymal stem cells. The differentiation can be inhibited by androgens, although knowledge about intracellular effectors of this inhibition is scarce. Recently, androgen-regulated microRNAs were detected as interesting candidates in this context. In this study, we analyse the role of miR-130a and miR-301 in the adipogenesis of human SGBS preadipocytes and whether they are prone to androgen regulation. Materials and Methods: microRNA expression during adipogenic differentiation with or without androgen stimulation was measured by qPCR. Putative target genes of miR-130a and miR-301 were identified by target database search and validated in luciferase reporter assays. Results: miR-130a and miR-301 are both significantly downregulated on day 3 and day 5 of adipogenic differentiation in comparison to day 0. Under androgen stimulation, a significant upregulation of miR-130a was detected after 7 days of adipogenesis lasting to day 14, while miR-301 did not change significantly until day 14. Luciferase reporter assays revealed the androgen receptor (AR), adiponectin (ADIPOQ) and tumour necrosis factor alpha (TNFα) as miR-130a target genes. Conclusions: miR-130a is an androgen-regulated microRNA that is downregulated during the early phase of adipogenesis and exerts its functions by regulating AR and ADIPOQ translation. These data may help to identify new signalling pathways associated with the androgen-mediated inhibition of adipogenesis.

Histological and molecular characterization of TFEB-rearranged renal cell carcinomas.

The 2016 WHO Classification of Tumors of the Urinary System recognizes microphthalmia transcription factor (MiT) family translocation carcinomas as a separate entity among renal cell carcinomas. TFE3 and transcription factor EB (TFEB) are members of the MiT family for which chromosomal rearrangements have been associated with renal cell carcinoma formation. TFEB translocation renal cell carcinoma is a rare tumor harboring a t(6;11)(p21;q12) translocation. Recently, renal cell carcinomas with TFEB amplification have been identified. TFEB amplified renal cell carcinomas have to be distinguished from TFEB-translocated renal cancer, because they may demonstrate a more aggressive behavior. Herein, we present a TFEB-translocated and a TFEB-amplified carcinoma cases and describe their distinct histological, immunohistochemical, and molecular characteristics. In addition, we review conventional morphology, immunophenotype, genetic background, and clinical outcome of TFEB-rearranged RCCs in the literature, with a special emphasis on important differential diagnoses and the diagnostic approach.

Semi-automated extraction and characterization of Stromal Vascular Fraction using a new medical device.

INTRODUCTION: The stem cell rich Stromal Vascular Fraction (SVF) can be harvested by processing lipo-aspirate or fat tissue with an enzymatic digestion followed by centrifugation. To date neither a standardised extraction method for SVF nor a generally admitted protocol for cell application in patients exists. A novel commercially available semi-automated device for the extraction of SVF promises sterility, consistent results and usability in the clinical routine. The aim of this work was to compare the quantity and quality of the SVF between the new system and an established manual laboratory method. MATERIAL AND METHODS: SVF was extracted from lipo-aspirate both by a prototype of the semi-automated UNiStation™ (NeoGenesis, Seoul, Korea) and by hand preparation with common laboratory equipment. Cell composition of the SVF was characterized by multi-parametric flow-cytometry (FACSCanto-II, BD Biosciences). The total cell number (quantity) of the SVF was determined as well the percentage of cells expressing the stem cell marker CD34, the leucocyte marker CD45 and the marker CD271 for highly proliferative stem cells (quality). RESULTS: Lipo-aspirate obtained from six patients was processed with both the novel device (d) and the hand preparation (h) which always resulted in a macroscopically visible SVF. However, there was a tendency of a fewer cell yield per gram of used lipo-aspirate with the device (d: 1.1×105±1.1×105 vs. h: 2.0×105±1.7×105; p = 0.06). Noteworthy, the percentage of CD34+ cells was significantly lower when using the device (d: 57.3% ±23.8% vs. h: 74.1% ±13.4%; p = 0.02) and CD45+ leukocyte counts tend to be higher when compared to the hand preparation (d: 20.7% ±15.8% vs. h: 9.8% ±7.1%; p = 0.07). The percentage of highly proliferative CD271+ cells was similar for both methods (d:12.9% ±9.6% vs. h: 13.4% ±11.6%; p = 0.74) and no differences were found for double positive cells of CD34+/CD45+ (d: 5.9% ±1.7% vs. h: 1.7% ±1.1%; p = 0.13), CD34+/CD271+ (d: 24.1% ±12.0% vs. h: 14.2% ±8.5%; p = 0.07). DISCUSSION: The semi-automated closed system provides a considerable amount of sterile SVF with high reproducibility. Furthermore, the SVF extracted by both methods showed a similar cell composition which is in accordance with the data from literature. This semi-automated device offers an opportunity to take research and application of the SVF one step further to the clinic.

Inhibition of adipogenic differentiation of human SGBS preadipocytes by androgen-regulated microRNA miR-375.

Late-onset hypogonadism (LOH), defined as a combination of low serum testosterone (T) levels in combination with clinical signs and symptoms of androgen deficiency in ageing men, is nowadays a well-characterized disease. Testosterone therapy in males affected by hypogonadism leads to a significant decrease of fat mass. In humans, the exact molecular mechanism of T effects on inhibition of adipogenesis is still unknown. We hypothesized that specific microRNAs could be regulated by androgens which might cause an inhibition of adipogenic differentiation. To confirm this hypothesis, human mesenchymal stem cells and a preadipocyte cell line were differentiated into mature adipocytes and in parallel treated with testosterone and dihydrotestosterone. The expression level of miR-375 was upregulated during adipogenic differentiation and downregulated after androgen treatment. Furthermore, we could show that after androgen treatment the decreased expression of miR-375 led to increased expression levels of adiponectin receptor 2 (ADIPOR2) compared to untreated adipocytes. Moreover, inhibition of miR-375 also mediated a decreased adipogenic differentiation and increased ADIPOR2 expression levels. In summary, we identified miR-375 as an androgen regulated microRNA, which could play an important role for understanding the mechanism of the increase in visceral fat mass and the associated insulin resistance caused by testosterone deficiency.