Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma.

BackgroundIncreasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic).MethodsWe exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate.ResultsAfter functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright cells from baseline (P < 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P > 0.05).ConclusionsAnti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Elevation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)-like interleukin 1β-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)-promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)-targeted therapy. Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions. Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7-mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance. Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC.</p

Elevation of c-FLIP in Castrate-Resistant Prostate Cancer Antagonizes Therapeutic Response to Androgen Receptor–Targeted Therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)–like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)–promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)–targeted therapy.Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P &lt; 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P &lt; 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7–mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC. Clin Cancer Res; 18(14); 3822–33

Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis

10.1371/journal.pone.0069604PLoS ONE87-POLN

Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma.

BackgroundIncreasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic).MethodsWe exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate.ResultsAfter functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P &lt; 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright cells from baseline (P &lt; 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P &lt; 0.05), but enriched ALDHbright cells 2.5 - 2.8 fold (P &lt; 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P &lt; 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P &lt; 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P &gt; 0.05).ConclusionsAnti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations

Factors Determining the Risk of Inadvertent Retroviral Transduction of Male Germ Cells After In Utero Gene Transfer in Sheep

The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction. Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation

EGFR mutational analysis of lung adenocarcinoma, malignant melanoma and colorectal carcinoma – Sanger/q-PCR sequencing versus NGS.

<p>EGFR mutational analysis of lung adenocarcinoma, malignant melanoma and colorectal carcinoma – Sanger/q-PCR sequencing versus NGS.</p

BRAF and KRAS mutational analysis of lung adenocarcinoma, malignant melanoma and colorectal carcinoma by NGS LA: lung adenocarcinoma; MM: malignant melanoma; CRC: colorectal cancer.

<p>BRAF and KRAS mutational analysis of lung adenocarcinoma, malignant melanoma and colorectal carcinoma by NGS LA: lung adenocarcinoma; MM: malignant melanoma; CRC: colorectal cancer.</p