Determining programmed cell death ligand 1 expression in circulating tumor cells of patients with clear cell renal cell carcinoma and its correlation with response to programmed cell death protein 1 inhibitors.

OBJECTIVE: There is a great interest in determining whether the expression of the programmed cell death ligand 1 is correlated with the efficacy of immune checkpoint inhibitors in patients with clear cell renal cell carcinoma; however, primary tumor biopsies can only provide limited information. Therefore, we explored the expression of programmed cell death ligand 1 on circulating tumor cells, which is a potential predictor of therapeutic response. METHODS: Circulating tumor cells were isolated from 20 clear cell renal cell carcinoma patients based on cell surface markers targeting clear cell renal cell carcinoma using IsoFlux device, followed by identification according to cell morphology and immunofluorescence studies. Programmed cell death ligand 1 expression status and clinical correlations were also analyzed. RESULTS: Before treatment with programmed cell death protein 1 inhibitors, circulating tumor cells were detected in all patients, ranging from 1 to 22 (median 7), with 75% (15/20) of the patients having programmed cell death ligand 1 + circulating tumor cells. Circulating tumor cell programmed cell death ligand 1 expression did not correlate with the immunohistochemical staining of programmed cell death ligand 1 in primary tumors. During treatment with programmed cell death protein 1 inhibitors, the disease control rate was much higher in the patients harboring programmed cell death ligand 1 + circulating tumor cells (73%, 11/15) than others (20%, 1/5). We also found that changes in total circulating tumor cell numbers and programmed cell death ligand 1 + circulating tumor cell counts correlated well with the disease outcome. CONCLUSION: We showed that the presence of programmed cell death ligand 1 + circulating tumor cells before programmed cell death protein 1 inhibition treatment could be a prognosis predictive factor and that the dynamic changes in circulating tumor cell numbers may be used to monitor the therapeutic response. Our study confirms the possibility of programmed cell death ligand 1 + circulating tumor cell detection in clear cell renal cell carcinoma patients' blood samples, which can potentially be used as an individualized immunotherapy molecular biomarker for real-time exploration.

Circulating Tumor Cells Predict Response of Neoadjuvant Chemotherapy in Patients with Bladder Cancer: A Preliminary Study.

This study aimed to explore the existence of circulating tumor cells (CTCs) in patients with muscle-invasive bladder cancer (MIBC) and their predictive potential for response to neoadjuvant chemotherapy (NAC). From 33 blood samples of MIBC patients, CTCs were isolated by cell surface markers and enriched by the IsoFlux™ device, followed by morphological and immunofluorescent identification. CTCs were detected at baseline in all samples. Immunofluorescence confirmed the tumor origin. MIBC patients were stratified by NAC response into the disease control (DC) and progressive disease (PD) groups. In the DC group, the number of CTCs decreased significantly after four courses of NAC (p < 0.0001). CTC counts in 7.5 mL after four NAC cycles were highly correlated with postoperative pathological T stage (p < 0.0001). Our study demonstrated that CTCs might represent a valuable predictive marker for NAC response in MIBC. CTC detection in MIBC patients could allow early arrangement of radical cystectomy for NAC non-responders to prevent disease progression while receiving the NAC courses.

Phase I Targeted Combination Trial of Sorafenib and GW5074 in Patients with Advanced Refractory Solid Tumors.

BACKGROUND: Combination therapy with the administration of GW5074 and sorafenib significantly induced necrotic death in various cancer cells in vivo, as well as prolonging the survival of an animal disease model due to significant suppression of the primary and metastatic lesions. We sought to determine the safety, tolerability, pharmacokinetics, and anti-tumor activity of this co-administration therapy in patients with refractory advanced solid cancers. METHODS: Twelve patients were enrolled. Eligible subjects received different dosages of GW5074 in one of the three dose cohorts (Cohort 1: 750 mg daily, Cohort 2: 1500 mg daily, Cohort 3: 750 mg twice daily) plus 200 mg of sorafenib daily to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) at phase 1. Furthermore, the expression level of phosphorylated DAPKS308 in primary tumor, metastatic tumor, and circulating tumor cells (CTC) were evaluated to investigate the relationship between biomarker and the efficacy profile. RESULTS: Among the 12 enrolled patients in this phase 1 trial, most adverse effects (AE) were grade 1, with two being grade 3. The most frequent AE of all grades were weight loss and hypertension, occurring in 16.7% of participants. Eight patients (66.7%) had the disease controlled by receiving co-administration therapy of GW5074 and sorafenib. GW5074 was found to have poor absorption, as increasing the dosage did not result in a significant increase in the bioavailability of GW5074 in subjects. Furthermore, the expression level of phosphorylated DAPKS308 in tumor and CTCs were correlated with the disease control rate (DCR) and duration of response (DOR). CONCLUSIONS: Co-administration therapy of GW5074 and sorafenib demonstrated a favorable safety profile and showed anti-tumor activity in a variety of tumor types. However, the solubility of GW5074 is not satisfactory. A future phase 2a trial will be carried out using the new salted form that has been proven to be more effective.

Detection of circulating tumor cells as therapeutic markers in patients with penile squamous cell carcinoma: A preliminary study.

BACKGROUND: This study aimed to investigate the presence of circulating tumor cells (CTCs) in patients with penile squamous cell carcinoma (PSCC). METHODS: CTCs were isolated from 14 patients with PSCC, 6 patients with balanoposthitis, and 6 healthy individuals. CTCs were enriched based on cell surface markers and filtered through the IsoFlux device, followed by identification according to cell morphology and immunofluorescence studies. RESULTS: CTCs were found in all PSCC blood samples but not in balanoposthitis samples and samples from healthy individuals. Immunofluorescence studies confirmed the tumor origin. When the patients with PSCC were stratified according to metastatic inguinal lymph node status, a statistically significant difference was observed in the number of detected CTCs. CONCLUSION: Our study showed that CTCs in PSCC may represent a valuable marker for differentiating PSCC from other tumors. Based on the correlation with some clinical parameters, CTC analysis is possibly relevant for noninvasive monitoring of disease progression and prognosis. The results also suggested a potential role of CTCs in preventing overtreatment, such as inguinal lymph node dissection.

Programmed Cell Death Ligand 1 Expression in Circulating Tumor Cells as a Predictor of Treatment Response in Patients with Urothelial Carcinoma.

Programmed cell death ligand 1 (PD-L1) inhibitors are commonly used in treating advanced-stage urothelial carcinoma (UC). Therefore, this study evaluated the relationship between PD-L1 expression in circulating tumor cells (CTCs) and treatment response to PD-L1 inhibitors using blood samples collected from patients with UC (n = 23). Subsequently, PD-L1 expression and its clinical correlation were analyzed. All patients had CTCs before PD-L1 inhibitory treatment, of which 15 had PD-L1-positive CTCs. However, PD-L1-positive expression in CTCs was not correlated with PD-L1 expression in tumor biopsy samples. Patients with PD-L1-positive CTCs had better disease control (DC) rates than those without PD-L1-positive CTCs. Moreover, changes in the proportion of PD-L1-positive CTCs were associated with disease outcomes. Furthermore, the PD-L1-positive CTC count in 9 of 11 patients who achieved DC had significantly decreased (p = 0.01). In four patients with progressive disease, this was higher or did not change. PD-L1-positive CTCs at baseline could be used as a biomarker to identify patients suitable for PD-L1 blockade therapy. Dynamic changes in PD-L1-positive CTCs during the course of treatment are predictive factors of immunotherapy response and prognostic factors of disease control. Hence, PD-L1-positive CTCs could be employed as a real-time molecular biomarker for individualized immunotherapy.

Effects of robot-assisted versus hand-assisted nephroureterectomy on circulating tumor cells for upper urinary tract urothelial carcinoma.

To compare perioperative circulating tumor cells (CTC) in primary upper tract urothelial carcinoma (UTUC) patients who underwent hand-assisted retroperitoneoscopic nephroureterectomy (HANU) or robotic-assisted nephroureterectomy (RANU). A total of 29 patients received RANU (n = 10) or HANU (n = 19). Peripheral blood samples were collected before, 24 h after surgery (POh24) and on postoperative day 28 (POD28). The demographic and pathologic data are similar in both groups. RANU had a longer operative time (p = 0.031), less bleeding volume (p = 0.004), and comparable pain sore (p = 0.169). The mean CTC numbers before surgery (2.4 vs. 2.3, p = 0.482), POh24 (2.4 vs. 1.9, p = 0.668) and POD28 (0.5 vs. 0.6, p = 0.280) were not significant different among groups. The amount of CTCs in both groups decreased and reached similar level on POD28. No significant difference of overall and intravesical recurrence rate between the two approaches. In comparison to RANU, more surgical manipulation does not affect tumor cell translocation into the bloodstream in UTUC patients who received HANU. However, a longer follow-up would be needed for the final comparison of tumor recurrence.

Hypoxia Stimulates the Epithelial-to-Mesenchymal Transition in Lung Cancer Cells Through Accumulation of Nuclear ß-Catenin.

BACKGROUND/AIM: Recent studies implied a significant role of hypoxia-inducible factor-1α (HIF1α) in cell transformation. This study aimed to assess the effects of HIF1α on the epithelial-to-mesenchymal transition (EMT) and tumorigenesis of lung adenocarcinoma cells. MATERIALS AND METHODS: Invasion, migration and colony formation assays were used to evaluate cell transformation. Expression of EMT-related markers were analyzed by western blot, reverse-transcription polymerase chain reaction or zymography. A luciferase assay was carried out to access the transcriptional activity of ß-catenin. RESULTS: Hypoxia enhanced migration, invasion and transformation of A549 lung adenocarcinoma cells. Hypoxic stimulation promoted the expression of EMT-related markers in lung cancer cells. The expression of HIF1α was found to be involved in hypoxia-mediated modulation of expression of snail family transcriptional repressors 1 (SNAI1) and 2 (SLUG). Hypoxia enhanced nuclear accumulation and transcriptional activity of ß-catenin. CONCLUSION: ß-Catenin promotes expression of EMT-related genes and eventually contributes to the metastatic process.

Impact of Enhancer of Zeste Homolog 2 on T Helper Cell-Mediated Allergic Rhinitis.

Enhancer of zeste homolog 2 (Ezh2) has been shown to play a role in the differentiation of T helper (Th) 1 and 2 cells in mice studies using Ezh2-deficient T cells. However, the results have been inconsistent, and the function of Ezh2 in human Th1 and Th2 cell differentiation and its association with disease remains controversial. We measured the expression of Ezh2 in Th1 and Th2 cells in peripheral blood mononuclear cells after acute challenge with house dust mite using flow cytometry in patients with allergic rhinitis (AR) and controls. The role of Ezh2 was further explored by adding the p38 inhibitor to see if this affected allergen-induced Th1 and Th2 differentiation. The expression of Ezh2 in the Th1 and Th2 cells was significantly lower in the patients than in the controls and was negatively correlated with serum IL-17A levels in the patients. Ex vivo allergen challenge resulted in rapid Th2 cell differentiation, which was negatively associated with the Ezh2 expression in Th2 cells. Inhibiting p38 activity increased the expression of Ezh2 in Th2 cells and reduced the number of differentiated Th2 cells. Our findings suggest that Ezh2 expression is potentially associated with AR development.

HAF mediates the evasive resistance of anti-angiogenesis TKI through disrupting HIF-1α and HIF-2α balance in renal cell carcinoma.

Anti-angiogenesis has emerged as a standard of care for metastatic renal cell carcinoma. However, long-lasting efficacy is seldom reached, and evasive resistance eventually occurs under anti-angiogenic tyrosine kinase inhibitor (TKI) therapy. To establish new therapeutic strategies, investigating the molecular mechanism of resistance is critically important. In our study, human umbilical vascular endothelial cells (HUVECs) were incubated with TKI treatment in conditioned medium derived from renal cancer cells (RCCs) to demonstrate cell viability. Quantitative real time PCR or Western blotting analysis detected the fluctuation of transcriptional factors HIF-1α and HIF-2α in RCCs under TKI treatment. We demonstrated the alteration of a specific cytokine produced from RCCs under normoxia or hypoxia incubation by utilizing a cytokine RT-PCR primer array. We found that the anti-angiogenic TKI sunitinib disrupted the balance between HIF-1α and HIF-2α in RCCs and led to a protective effect on HUVECs against sunitinib treatment when cultured with conditioned medium. Mechanistically, RCCs treated with sunitinib resulted in down-regulation of HIF-1α, but not HIF-2α, through reduction of both mRNA and protein levels. The down-regulation of HIF-1α by sunitinib occurred via hypoxia associated factor (HAF), which also enhanced HIF-2α transactivation activity to increase the production of pro-angiogenic factors and cytokines and promote HUVEC proliferation. This phenomenon was observed in ACHN and A498 cells, which express both HIF-1α and HIF-2α, but was not observed in 786-O cells, which express only HIF-2α. Our results illustrated that targeting both angiogenesis and hypoxia pathways might provide a resolution to dealing with the devastating effects of anti-angiogenesis resistance.

Novel Cancer Therapeutics with Allosteric Modulation of the Mitochondrial C-Raf-DAPK Complex by Raf Inhibitor Combination Therapy.

Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf(S338)) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK(S308)), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF(C-Raf-/-) or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK(S308), this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf(S338) and pDAPK(S308) translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK(S308) to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK(S308) modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation.

Pharmacologic down-regulation of EZH2 suppresses bladder cancer in vitro and in vivo.

The polycomb group gene, EZH2, is highly expressed in advanced bladder cancer. Here we demonstrated that down-regulation of EZH2 in tumor tissues after neo-adjuvant chemotherapy correlated with good therapeutic response in advanced bladder cancer. We next developed a small molecule, NSC745885, derived from natural anthraquinone emodin, which down-regulated EZH2 via proteasome-mediated degradation. NSC745885 showed potent selective toxicity against multiple cancer cell lines but not normal cells. NSC745885 treatment overcame multiple-drug resistance and inhibited growth of resistant cancer cells. Over-expression of EZH2 in cancer cells attenuated effects of NSC745885, suggesting that down-regulation of EZH2 was responsible for growth inhibition of NSC745885. NSC745885 also suppressed tumor growth and down-regulated EZH2 in vivo. These results indicate that NSC7455889 suppresses bladder cancer by targeting EZH2.

Effect of neuromuscular electrical stimulation in a patient with Sjogren's syndrome with dysphagia: a real time videofluoroscopic swallowing study.

Severe dysphagia in a 54 year-old woman with Sjogren's syndrome with involvement of multiple cranial nerves significantly improved after treatment with neuromuscular electrical stimulation (NMES) in combination with a swallowing rehabilitation program. The swallowing response was assessed in real time using a videofluoroscope. Immediate improvement in the tongue retraction force, clearing of the valleculae, increase in laryngeal elevation and shortening of pharyngeal transit time were noted during stimulation. The patient returned to independent oral feeding after 46 sessions of NMES. After follow-up for 1 year, we found that the patient maintained adequate oral feeding and did not show signs of pulmonary complications.

The electromyographic responses of paraspinal muscles during isokinetic exercise in adolescents with idiopathic scoliosis with a Cobb's angle less than fifty degrees.

BACKGROUND: Analysis of electromyographic (EMG) activities in the back during dynamic exercise is needed because more complex loading on the spine is created in comparison with that during static exercise. The purpose of this study is to investigate the difference in bilateral midback and low-back paraspinal muscle (PSM) activities during performance of different resistance isokinetic exercises in healthy subjects and those with scoliosis. METHODS: Forty-one healthy subjects and thirty-three subjects with adolescent idiopathic scoliosis (AIS) were enrolled. An isokinetic back system in combination with quantitative surface EMG was used to evaluate the dominant and nondominant PSM activities by analyzing the root mean square (RMS) during isokinetic extension and flexion exercise at velocities of 30 degree/s and 90 degree/s. RESULTS: Significantly higher RMS of EMG were found in the dominant medial and lateral PSM of the lumbar region than the non-dominant muscles in the healthy control group and in those with AIS with smaller curves (<20 degrees) during isokinetic flexion and extension exercises. In AIS patients with larger curves (20 to 50 degrees), shifting of muscle activities from the dominant to the non-dominant side occurred during isokinetic exercises, and the EMG activities of the thoracic muscle were significantly higher on the non-dominant (concave) side than on the dominant (convex) side. CONCLUSIONS: The bilateral PSM do not act symmetrically during isokinetic back exercises. The dominant lumbar PSM supply the major action in healthy subjects and patients with small curve scoliosis. For larger curve scoliosis, compensated muscle activity is needed in the midback when doing resistance exercises. More midback protection may be needed by scoliotic subjects with large curves during resistance exercise.