Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

Introduction: The prognosis of glioblastoma (GBM) treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ)/radiotherapy remains very poor (less than 15 months). GBMs have been found to contain a small population of cancer stem cells (CSCs) that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID) that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care. Methods: Forty-one GBM patients (mean age 54 years, 59% male), all eligible for a surgical biopsy, were enrolled in an Institutional Review Board–approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS), and overall survival (OS). Odds ratio (OR) associations of 12-month recurrence, PFS, and OS outcomes were estimated for CSC, bulk tumor, and combined assay responses for the standard-of-care TMZ treatment; sensitivities/specificities, areas under the curve (AUCs), and risk reclassification components were examined. Results: Median follow-up was 8 months (range 3-49 months). For every 5% increase in in vitro CSC cell kill by TMZ, 12-month patient response (nonrecurrence of cancer) increased two-fold, OR = 2.2 (P = .016). Similar but somewhat less supported associations with the bulk tumor test were seen, OR = 2.75 (P = .07) for each 5% bulk tumor cell kill by TMZ. Combining CSC and bulk tumor assay results in a single model yielded a statistically supported CSC association, OR = 2.36 (P = .036), but a much attenuated remaining bulk tumor association, OR = 1.46 (P = .472). AUCs and [sensitivity/specificity] at optimal outpoints (>40% CSC cell kill and >55% bulk tumor cell kill) were AUC = 0.989 [sensitivity = 100/specificity = 97], 0.972 [100/89], and 0.989 [100/97] for the CSC only, bulk tumor only, and combined models, respectively. Risk categorization of patients was improved by 11% when using the CSC test in conjunction with the bulk test (risk reclassification nonevent net reclassification improvement [NRI] and overall NRI = 0.111, P = .030). Median recurrence time was 20 months for patients with a positive (>40% cell kill) CSC test versus only 3 months for those with a negative CSC test, whereas median recurrence time was 13 months versus 4 months for patients with a positive (>55% cell kill) bulk test versus negative. Similar favorable results for the CSC test were observed for PFS and OS outcomes. Panel results across 14 potential other treatments indicated that 34/41 (83%) potentially more optimal alternative therapies may have been chosen using CSC results, whereas 27/41 (66%) alternative therapies may have been chosen using bulk tumor results. Conclusions: The ChemoID CSC drug response assay has the potential to increase the accuracy of bulk tumor assays to help guide individualized chemotherapy choices. GBM cancer recurrence may occur quickly if the CSC test has a low in vitro cell kill rate even if the bulk tumor test cell kill rate is high.     

Turning Cancer Stem Cells Inside Out: An Exploration of Glioma Stem Cell Signaling Pathways

Tumors are complex collections of heterogeneous cells with recruited vasculature, inflammatory cells, and stromal elements. Neoplastic cells frequently display a hierarchy in differentiation status. Recent studies suggest that brain tumors have a limited population of neoplastic cells called cancer stem cells with the capacity for sustained self-renewal and tumor propagation. Brain tumor stem cells contribute to therapeutic resistance and tumor angiogenesis. In this minireview, we summarize recent data regarding critical signaling pathways involved in brain tumor stem cell biology and discuss how targeting these molecules may contribute to the development of novel anti-glioma therapies.Cancers can be considered organ systems with aberrant activation of developmental and wound response pathways. Recent evidence suggests that within some tumors there is a cell subpopulation with the special capacity for sustained self-renewal and tumor propagation in vivo. Cells fulfilling these criteria were originally reported in acute myeloid leukemia (1), but similar populations were soon successively identified within various solid tumors (2). The proper terminology regarding these cells remains unsettled, with most groups using terms such as CSCs,² tumor-initiating/propagating cells, and stem-like cancer cells. Although CSCs are a source of controversy, the concept recognizes the well described heterogeneity of tumor cells. Many critics contest the hypothesis on the grounds of a potential stem cell origin, challenge of current markers, or CSC frequency, none of which are implicit requirements of the CSC hypothesis (3).Malignant gliomas are essentially universally lethal despite conventional therapy, with surgical resection and chemoradiation limited to palliation. Glioma CSCs were among the first solid tumor CSCs described (4) and remain one of the most widely used CSC models. Glioma CSCs share significant similarities with normal NSCs, including the expression of stem cell markers (CD133, Nestin, Musashi, and Sox2) and the capacity to differentiate into multiple lineages (5), but the overlap is incomplete. Notably, glioma CSCs are also highly resistant to chemoradiotherapies (5, 6), underscoring the importance of developing more efficient therapies against CSCs and prompting researchers to elucidate the molecular mechanisms regulating CSCs. Here, we summarize recent findings regarding the signaling pathways that are critical to glioma CSC biology.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

Isolation of Cancer Stem Cells from Three Human Glioblastoma Cell Lines: Characterization of Two Selected Clones

Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca²⁺ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca²⁺-channels but they exhibited increased intracellular Ca²⁺ levels in response to ATP. These Ca²⁺ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca²⁺-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.     

Longitudinal Treatment Outcomes of Microsurgical Treatment of Neurosensory Deficit after Lower Third Molar Surgery: A Prospective Case Series

Objective

To prospectively evaluate the longitudinal subjective and objective outcomes of the microsurgical treatment of lingual nerve (LN) and inferior alveolar nerve (IAN) injury after third molar surgery.

Materials and Methods

A 1-year longitudinal observational study was conducted on patients who received LN or IAN repair after third molar surgery-induced nerve injury. Subjective assessments (“numbness”, “hyperaesthesia”, “pain”, “taste disturbance”, “speech” and “social life impact”) and objective assessments (light touch threshold, two-point discrimination, pain threshold, and taste discrimination) were recorded.

Results

12 patients (10 females) with 10 LN and 2 IAN repairs were recruited. The subjective outcomes at post-operative 12 months for LN and IAN repair were improved. “Pain” and “hyperaesthesia” were most drastically improved. Light touch threshold improved from 44.7g to 1.2g for LN repair and 2g to 0.5g for IAN repair.

Conclusion

Microsurgical treatment of moderate to severe LN injury after lower third molar surgery offered significant subjective and objective sensory improvements. 100% FSR was achieved at post-operative 6 months.     

Prognostic Value of Cancer Stem Cell Marker Aldehyde Dehydrogenase in Ovarian Cancer: A Meta-Analysis

Objective

Aldehyde dehydrogenase (ALDH) has recently been reported as a marker of cancer stem-like cells in ovarian cancer. However, the prognostic role of ALDH in ovarian cancer still remains controversial. In this study, we aimed to evaluate the association between the expression of ALDH and the outcome of ovarian cancer patients by performing a meta-analysis.

Methods

We systematically searched for studies investigating the relationships between ALDH expression and outcome of ovarian cancer patients. Only articles in which ALDH expression was detected by immunohistochemical staining were included. A meta-analysis was performed to generate combined hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS) and disease-free survival (DFS).

Results

A total of 1,258 patients from 7 studies (6 articles) were included in the analysis. Our results showed that high ALDH expression in patients with ovarian cancer was associated with poor prognosis in terms of Os (HR, 1.25; 95% CI, 1.07-1.47; P = 0.005) and DFS (HR, 1.58; 95% CI, 1.00-2.49; P = 0.052), though the difference for DFS was not statistically significant. In addition, there was no evidence of publication bias as suggested by Begg’s and Egger’s tests (Begg’s test, P = 0.707; Egger’s test, P = 0.355).

Conclusion

The present meta-analysis indicated that elevated ALDH expression was associated with poor prognosis in patients with ovarian cancer.     

Cancer Stem Cells Sensitivity Assay (STELLA) in Patients with Advanced Lung and Colorectal Cancer: A Feasibility Study

Background

Cancer stem cells represent a population of immature tumor cells found in most solid tumors. Their peculiar features make them ideal models for studying drug resistance and sensitivity. In this study, we investigated whether cancer stem cells isolation and in vitro sensitivity assay are feasible in a clinical setting.

Methods

Cancer stem cells were isolated from effusions or fresh cancer tissue of 23 patients who progressed after standard therapy failure. Specific culture conditions selected for immature tumor cells that express markers of stemness. These cells were exposed in vitro to chemotherapeutic and targeted agents.

Results

Cancer stem cells were extracted from liver metastases in 6 cases (25%), lung nodules in 2 (8%), lymph node metastases in 3 (12.5%) and pleural/peritoneal/pericardial effusion in 13 (54%). Cancer stem cells were successfully isolated in 15 patients (63%), including 14 with lung cancer (93.3%). A sensitivity assay was successfully performed in 7 patients (30.4%), with a median of 15 drugs/combinations tested (range 5-28) and a median time required for results of 51 days (range 37-95).

Conclusion

The approach used for the STELLA trial allowed isolation of cancer stem cells in a consistent proportion of patients. The low percentage of cases completing the full procedure and the long median time for obtaining results highlights the need for a more efficient procedure.

Trial Registration

ClinalTrials.gov NCT01483001     

The Role of a Single Angiogenesis Inhibitor in the Treatment of Recurrent Glioblastoma Multiforme: A Meta-Analysis and Systematic Review

Background

Currently, the standard treatment for newly diagnosed glioblastoma multiforme (GBM) is maximal safe surgical resection followed by radiation therapy with concurrent and adjuvant temozolomide. However, disease recurs in almost all patients, and the optimal salvage treatment for recurrent GBM remains unclear. We conducted a systematic review and meta-analysis of published clinical trials to assess the efficacy and toxicities of angiogenesis inhibitors alone as salvage treatment in these patients.

Methods

Trials published between 1994 and 2015 were identified by an electronic search of public databases (MEDLINE, EMBASE, Cochrane library). Demographic data, treatment regimens, objective response rate (ORR), median progression-free survival (PFS), median overall survival (OS), 6-months PFS rate, 1-year OS and grade 3/4 toxicities were extracted. We also compared the main outcomes of interest between bevacizumab and other angiogenesis inhibitors. All analyses were performed using Comprehensive Meta Analysis software (Version 2.0).

Results

A total of 842 patients were included for analysis: 343 patients were treated with bevacizumab, 386 with other angiogenesis inhibitors and 81 with thalidomide. The pooled ORR, 6-months PFS, and 1-year OS for recurrent GBM patients receiving angiogenesis inhibitors was 20.1%, 19.5% and 29.3%, respectively. The use of single agent bevacizumab in recurrent GBM significantly improved ORR and 6-months PFS when compared to other angiogenesis inhibitors [relative risk (RR) 2.93, 95% CI 1.38–6.21; p = 0.025; and RR 2.36 95% CI 1.46–3.82; p<0.001, respectively], while no significant difference in 1-year OS was found between the two groups (p = 0.07). when compared to thalidomide, bevacizumab treatment in recurrent GBM significantly improved ORR (RR 6.8, 95%CI: 2.64–17.6, p<0.001), but not for 6-months PFS (p = 0.07) and 1-year OS (p = 0.31). As for grade 3/4 toxicities, the common toxicity was hypertension with pooled incidence of 12.1%, while high-grade thromboembolic events (2.2%), hemorrhage (5.1%) and GI perforation (2.8%) associated with angiogenesis inhibitors were relatively low.

Conclusions

In comparison with other angiogenesis inhibitors and thalidomide, the use of single agent bevacizumab as salvage treatment for recurrent GBM patients improve ORR and 6-months PFS, but not for 1-year OS.     

Teriparatide as a Systemic Treatment for Lower Extremity Nonunion Fractures: A Case Series

ObjectiveTo investigate the effect of teriparatide (parathyroid hormone [1-34]) on the healing of long bone nonunion fractures.MethodsWe performed a retrospective chart review of patients with fracture nonunion, aged 10 to 99 years who were treated with teriparatide at the Children’s Hospital of Philadelphia or the Hospital of the University of Pennsylvania between November 2002 and January 2013. The primary endpoints were radiographic evidence of callus formation and fracture union, ability to bear weight without affected limb limp, and normal range of motion and strength.ResultsSix patients aged 19 to 64 years with tibial or femoral fractures that had not healed for 3 to 36 months were treated with teriparatide 20 μg/day. Accelerated healing of fracture nonunion was confirmed in 5 of 6 patients with time to complete union of 3 to 9 months. The shortest time to recovery was observed in younger patients without comorbidities. Treatment was well tolerated.ConclusionTeriparatide is a promising treatment for nonunion fractures, but its response depends on associated comorbidities. The potential benefit of teriparatide as an adjunct to treat nonunion justifies randomized placebo-controlled trials to determine its efficacy and safety in broader populations. (Endocr Pract. 2015;21:136-142)     

Prognostic Value of Cancer Stem Cell Marker ALDH1 Expression in Colorectal Cancer: A Systematic Review and Meta-Analysis

Objective

Many studies have indicated the prognostic and clinicopathological value of aldehyde dehydrogenase 1 (ALDH1) in colorectal cancer (CRC) patients still remains controversial. Thus we performed this study to clarify the relationship between high ALDH1 expression in CRC and its impact on survival and clinicopathological features.

Methods

Publications for relevant studies in Pubmed, the Cochrane Library, Embase, and China National Knowledge Infrastructure (CNKI) through April 2015 were identified. Only articles describing ALDH1 antigen with immunohistochemistry in CRC were included. The software RevMan 5.1 was used to analyze the outcomes, including 5-year overall survival (OS), disease-free survival (DFS) and clinicopathological features.

Results

9 studies with 1203 patients satisfying the criteria were included. The overall rate of high ALDH1 expression was 46.5% by immunohistochemical staining. High ALDH1 expression as an independent prognostic factor was significantly associated with the 5-year OS and DFS (OR = 0.42, 95%CI: 0.26–0.68, P = 0.0004; OR = 0.38, 95%CI: 0.24–0.59, P < 0.0001, respectively). High ALDH1 expression was highly correlated with the tumor (T) stage (T3 + T4 vs. T1 + T2; OR = 2.16, 95%CI: 1.09–4.28, P = 0.03), lymph node (N) stage (N1 + N2 vs. N0; OR = 1.8; 95%CI: 1.17–2.79, P = 0.008), and tumor differentiation (G3 vs. G1 + G2; OR = 1.88; 95%CI: 1.07–3.30, P = 0.03). However, high ALDH1 expression was not significantly correlated with the patient age (>60 years old vs. <60 years old; OR = 1.11, 95%CI: 0.63–1.94, P = 0.72).

Conclusions

High ALDH1 expression indicates a poor prognosis in CRC patients. Moreover, high ALDH1 expression correlates with the T stage, N stage, and tumor differentiation, but not with age.     

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.     

慢性髓性白血病患者非血缘供者骨髓移植术后并发甲状腺功能亢进1例并文献复习

目的:分析骨髓移植后引起甲状腺功能亢进的病因、临床特点、治疗及预后.方法:报道l例慢性髓性白血病患者非血缘供者骨髓移植术后并发甲状腺功能亢进随访9年的结果,并复习相关文献.结果:患者男性,21岁,确诊慢性髓性白血病后行非血缘供者骨髓移植,术后+44天,出现持续低热,抗细菌、抗病毒及抗真菌治疗均无效,排除疾病复发,查甲状腺功能提示患者从亚临床甲状腺功能亢进进展为甲状腺功能亢进,给予口服甲巯咪唑治疗2周,患者体温降至正常,血T4、fT4恢复正常,随访9年,慢性髓性白血病无复发,甲状腺功能持续正常.检索文献发现类似报道5例,对其进行归纳分析.结论:骨髓移植术后早期并发甲状腺功能亢进,可能与放化疗预处理及免疫损伤引起的破坏性甲状腺炎相关,有其独特的临床表现,极易被忽视,治疗方面可以尝试应用糖皮质激素及抗甲状腺药物.对于非血缘供者骨髓移植后不明原因发热者,应考虑到甲亢可能.     

Intranasal Mesenchymal Stem Cell Treatment for Neonatal Brain Damage: Long-Term Cognitive and Sensorimotor Improvement

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5×10⁶ MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.     

Lucid Dreaming Treatment for Nightmares: A Series of Cases

Goal of this series of cases was to investigate lucid dreaming treatment for nightmares. Hypotheses were that lucid dreaming treatment would decrease nightmare frequency and state/trait anxiety, and improve the quality of sleep. Eight participants received a one-hour individual session, which consisted of lucid dreaming exercises and discussing possible constructive solutions for the nightmare. Nightmare frequency and sleep quality were measured by a sleep questionnaire, anxiety was measured by the Spielberger State and Trait Anxiety Inventory. At the follow-up two months later the nightmare frequency had decreased, while the sleep quality had increased slighty. There were no changes on state and trait anxiety. Lucid dreaming treatment seems to be effective in reducing nightmare frequency, although the effective factor remains unclear.