Growth in Virologically Suppressed HIV Positive Children on Antiretroviral Therapy: Individual and Population-Level References

BACKGROUND Combination antiretroviral therapy (ART) suppresses viral replication in HIV-infected children. The growth of virologically suppressed children on ART has not been well documented. We aimed to develop dynamic reference curves for weight-for-age z scores (WAZ) and height-for-age z scores (HAZ). RESULTS A total of 4,876 children were followed for 7,407 person-years. Analyses were stratified by baseline z-scores and age, which were the most important predictors of growth response. The youngest children showed the most pronounced increase in weight and height initially but catch-up growth stagnated after 1-2 years. Three years after starting ART, WAZ ranged from -2.2 (95% Prediction interval -5.6 to 0.8) in children with baseline age "5 years and z-score "-3 to 0.0 (-2.7 to 2.4) in children with baseline age "2 years and WAZ "-1. For HAZ the corresponding range was -2.3 (-4.9 to 0.3) in children with baseline age"5 years and z-score "-3 to 0.3 (-3.1 to 3.4) in children with baseline age 2-5 years and HAZ "-1. CONCLUSIONS We have developed an online tool to calculate reference trajectories in fully suppressed children. The web application could help to define 'optimal' growth response and identify children with treatment failure.

What is the influence of randomisation sequence generation and allocation concealment on treatment effects of physical therapy trials? A meta-epidemiological study.

OBJECTIVE To determine if adequacy of randomisation and allocation concealment is associated with changes in effect sizes (ES) when comparing physical therapy (PT) trials with and without these methodological characteristics. DESIGN Meta-epidemiological study. PARTICIPANTS A random sample of randomised controlled trials (RCTs) included in meta-analyses in the PT discipline were identified. INTERVENTION Data extraction including assessments of random sequence generation and allocation concealment was conducted independently by two reviewers. To determine the association between sequence generation, and allocation concealment and ES, a two-level analysis was conducted using a meta-meta-analytic approach. PRIMARY AND SECONDARY OUTCOME MEASURES association between random sequence generation and allocation concealment and ES in PT trials. RESULTS 393 trials included in 43 meta-analyses, analysing 44 622 patients contributed to this study. Adequate random sequence generation and appropriate allocation concealment were accomplished in only 39.7% and 11.5% of PT trials, respectively. Although trials with inappropriate allocation concealment tended to have an overestimate treatment effect when compared with trials with adequate concealment of allocation, the difference was non-statistically significant (ES=0.12; 95% CI -0.06 to 0.30). When pooling our results with those of Nuesch et al, we obtained a pooled statistically significant value (ES=0.14; 95% CI 0.02 to 0.26). There was no difference in ES in trials with appropriate or inappropriate random sequence generation (ES=0.02; 95% CI -0.12 to 0.15). CONCLUSIONS Our results suggest that when evaluating risk of bias of primary RCTs in PT area, systematic reviewers and clinicians implementing research into practice should pay attention to these biases since they could exaggerate treatment effects. Systematic reviewers should perform sensitivity analysis including trials with low risk of bias in these domains as primary analysis and/or in combination with less restrictive analyses. Authors and editors should make sure that allocation concealment and random sequence generation are properly reported in trial reports.

Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial

BACKGROUND Long-term hormone therapy has been the standard of care for advanced prostate cancer since the 1940s. STAMPEDE is a randomised controlled trial using a multiarm, multistage platform design. It recruits men with high-risk, locally advanced, metastatic or recurrent prostate cancer who are starting first-line long-term hormone therapy. We report primary survival results for three research comparisons testing the addition of zoledronic acid, docetaxel, or their combination to standard of care versus standard of care alone. METHODS Standard of care was hormone therapy for at least 2 years; radiotherapy was encouraged for men with N0M0 disease to November, 2011, then mandated; radiotherapy was optional for men with node-positive non-metastatic (N+M0) disease. Stratified randomisation (via minimisation) allocated men 2:1:1:1 to standard of care only (SOC-only; control), standard of care plus zoledronic acid (SOC + ZA), standard of care plus docetaxel (SOC + Doc), or standard of care with both zoledronic acid and docetaxel (SOC + ZA + Doc). Zoledronic acid (4 mg) was given for six 3-weekly cycles, then 4-weekly until 2 years, and docetaxel (75 mg/m(2)) for six 3-weekly cycles with prednisolone 10 mg daily. There was no blinding to treatment allocation. The primary outcome measure was overall survival. Pairwise comparisons of research versus control had 90% power at 2·5% one-sided α for hazard ratio (HR) 0·75, requiring roughly 400 control arm deaths. Statistical analyses were undertaken with standard log-rank-type methods for time-to-event data, with hazard ratios (HRs) and 95% CIs derived from adjusted Cox models. This trial is registered at ClinicalTrials.gov (NCT00268476) and ControlledTrials.com (ISRCTN78818544). FINDINGS 2962 men were randomly assigned to four groups between Oct 5, 2005, and March 31, 2013. Median age was 65 years (IQR 60-71). 1817 (61%) men had M+ disease, 448 (15%) had N+/X M0, and 697 (24%) had N0M0. 165 (6%) men were previously treated with local therapy, and median prostate-specific antigen was 65 ng/mL (IQR 23-184). Median follow-up was 43 months (IQR 30-60). There were 415 deaths in the control group (347 [84%] prostate cancer). Median overall survival was 71 months (IQR 32 to not reached) for SOC-only, not reached (32 to not reached) for SOC + ZA (HR 0·94, 95% CI 0·79-1·11; p=0·450), 81 months (41 to not reached) for SOC + Doc (0·78, 0·66-0·93; p=0·006), and 76 months (39 to not reached) for SOC + ZA + Doc (0·82, 0·69-0·97; p=0·022). There was no evidence of heterogeneity in treatment effect (for any of the treatments) across prespecified subsets. Grade 3-5 adverse events were reported for 399 (32%) patients receiving SOC, 197 (32%) receiving SOC + ZA, 288 (52%) receiving SOC + Doc, and 269 (52%) receiving SOC + ZA + Doc. INTERPRETATION Zoledronic acid showed no evidence of survival improvement and should not be part of standard of care for this population. Docetaxel chemotherapy, given at the time of long-term hormone therapy initiation, showed evidence of improved survival accompanied by an increase in adverse events. Docetaxel treatment should become part of standard of care for adequately fit men commencing long-term hormone therapy. FUNDING Cancer Research UK, Medical Research Council, Novartis, Sanofi-Aventis, Pfizer, Janssen, Astellas, NIHR Clinical Research Network, Swiss Group for Clinical Cancer Research.

Towards tailored radiopeptide therapy.

PURPOSE Somatostatin receptor-targeted radiopeptide therapy is commonly performed using single radioisotopes. We evaluated the benefits and harms of combining radioisotopes in radiopeptide therapy in patients with neuroendocrine tumor. METHODS Using multivariable-adjusted survival analyses and competing risk analyses we evaluated outcomes in patients with neuroendocrine tumor receiving (90)Y-DOTATOC, (177)Lu-DOTATOC or their combination. RESULTS (90)Y-DOTATOC plus (177)Lu-DOTATOC treatment was associated with longer survival than (90)Y-DOTATOC (66.1 vs. 47.5 months; n = 1,358; p < 0.001) or (177)Lu-DOTATOC alone (66.1 vs. 45.5 months; n = 390; p < 0.001). (177)Lu-DOTATOC was associated with longer survival than (90)Y-DOTATOC in patients with solitary lesions (HR 0.3, range 0.1 - 0.7; n = 153; p = 0.005), extrahepatic metastases (HR 0.5, range 0.3 - 0.9; n = 256; p = 0.029) and metastases with low uptake (HR 0.1, range 0.05 - 0.4; n = 113; p = 0.001). (90)Y-DOTATOC induced higher hematotoxicity rates than combined treatment (9.5% vs. 4.0%, p = 0.005) or (177)Lu-DOTATOC (9.5 vs. 1.4%, p = 0.002). Renal toxicity was similar among the treatments. CONCLUSIONS Using (90)Y and (177)Lu might facilitate tailoring radiopeptide therapy and improve survival in patients with neuroendocrine tumors.

Glenohumeral dislocation. Injury patterns and combination injuries

Hintergrund Begleitverletzungen können in bis zu 90 % der Fälle nach erstmaliger Schulterluxation auftreten. Auch wenn sie nicht immer einen Einfluss auf die Therapiewahl haben, so ist eine sorgfältige Diagnostik entscheidend. Einteilung In der Akutsituation ist eine konventionelle Bildgebung in mindestens 2 Ebenen (a.-p./Neer/evtl. axial) vor und nach Reposition zwingend. Luxationsfrakturen dürfen nicht übersehen bzw. durch das Manöver der geschlossenen Reposition sekundär disloziert werden. Bestehen ossäre glenoidale, humerale oder kombinierte Verletzungen, sollten sie gemäß Stabilitätskriterien versorgt werden. Dies kann umgehend, nach manifester Dezentrierung oder Instabilität entweder mittels Osteosythese oder als glenohumerale Stabilisation im Verlauf erfolgen. Bei einer Instabilität ist prinzipiell zur Bilanzierung einer ossären Ursache das Arthro-CT die Untersuchung der Wahl, welche auch eine Beurteilung der kapsulolabroligamentären Verletzung sowie einer traumatischen Rotatorenmanschettenläsion ermöglicht. Letztere ist jedoch besser mittels Arthro-MRT zu beurteilen. Diskussion Eine signifikante frische, meist größere oder massive, Rotatorenmanschettenläsion sollte rasch operativ angegangen werden. Medial reichende „off the track“ Hill-Sachs-Läsionen können mittels einer Hill-Sachs-Remplissage oder, wie auch glenoidale Defekte, mittels einer Kochenaugmentation versorgt werden. Langzeitresultate des Latarjet-Verfahrens zeigen 25 Jahre nach dem Eingriff die niedrigste Reluxationsrate < 4 %, eine gute Außenrotation, eine sehr hohe Patientenzufriedenheit und degenerative Veränderungen, welche vergleichbar mit der natürlichen Entwicklung nach erstmaliger Schulterluxation ohne Rezidiv sind.

EMAS position statement: Testosterone replacement therapy in the aging male‏.

INTRODUCTION Late-onset hypogonadism (LOH) represents a common clinical entity in aging males, characterized by the presence of symptoms (most usually of a sexual nature, such as decreased libido, decreased spontaneous erections and erectile dysfunction) and signs, in combination with low serum testosterone concentrations. Whether testosterone replacement therapy (TRT) should be offered to those individuals is still under extensive debate. AIMS The aim of this position statement is to provide and critically appraise evidence on TRT in the aging male, focusing on pathophysiology and characteristics of LOH, indications for TRT, available therapeutic agents, monitoring and treatment-associated risks. MATERIALS AND METHODS Literature review and consensus of expert opinion. RESULTS AND CONCLUSIONS Diagnosis and treatment of LOH is justified, if a combination of symptoms of testosterone deficiency and low testosterone is present. Patients receiving TRT could profit with regard to obesity, metabolic syndrome, type 2 diabetes mellitus, sexual function and osteoporosis and should undergo scheduled testing for adverse events regularly. Potential adverse effects of TRT on cardiovascular disease, prostate cancer and sleep apnea are as yet unclear and remain to be investigated in large-scale prospective studies. Management of aging men with LOH should include individual evaluation of co-morbidities and careful risk versus benefit assessment.

Robustness of the Voluntary Breath-Hold Approach for the Treatment of Peripheral Lung Tumors Using Hypofractionated Pencil Beam Scanning Proton Therapy.

PURPOSE The safe clinical implementation of pencil beam scanning (PBS) proton therapy for lung tumors is complicated by the delivery uncertainties caused by breathing motion. The purpose of this feasibility study was to investigate whether a voluntary breath-hold technique could limit the delivery uncertainties resulting from interfractional motion. METHODS AND MATERIALS Data from 15 patients with peripheral lung tumors previously treated with stereotactic radiation therapy were included in this study. The patients had 1 computed tomographic (CT) scan in voluntary breath-hold acquired before treatment and 3 scans during the treatment course. PBS proton treatment plans with 2 fields (2F) and 3 fields (3F), respectively, were calculated based on the planning CT scan and subsequently recalculated on the 3 repeated CT scans. Recalculated plans were considered robust if the V95% (volume receiving ≥95% of the prescribed dose) of the gross target volume (GTV) was within 5% of what was expected from the planning CT data throughout the simulated treatment. RESULTS A total of 14/15 simulated treatments for both 2F and 3F met the robustness criteria. Reduced V95% was associated with baseline shifts (2F, P=.056; 3F, P=.008) and tumor size (2F, P=.025; 3F, P=.025). Smaller tumors with large baseline shifts were also at risk for reduced V95% (interaction term baseline/size: 2F, P=.005; 3F, P=.002). CONCLUSIONS The breath-hold approach is a realistic clinical option for treating lung tumors with PBS proton therapy. Potential risk factors for reduced V95% are small targets in combination with large baseline shifts. On the basis of these results, the baseline shift of the tumor should be monitored (eg, through image guided therapy), and appropriate measures should be taken accordingly. The intrafractional motion needs to be investigated to confirm that the breath-hold approach is robust.

Secondary analysis of effectivness of tinidazole-containing-quadruple eradication therapy in asymptomatic H. pylori infected children in El Paso

Helicobacter pylori infection is frequently acquired during childhood. This microorganism is known to cause gastritis, and duodenal ulcer in pediatric patients, however most children remain completely asymptomatic to the infection. Currently there is no consensus in favor of treatment of H. pylori infection in asymptomatic children. The firstline of treatment for this population is triple medication therapy including two antibacterial agents and one proton pump inhibitor for a 2 week duration course. Decreased eradication rate of less than 75% has been documented with the use of this first-line therapy but novel tinidazole-containing quadruple sequential therapies seem worth investigating. None of the previous studies on such therapy has been done in the United States of America. As part of an iron deficiency anemia study in asymptomatic H. pylori infected children of El Paso, Texas, we conducted a secondary data analysis of study data collected in this trial to assess the effectiveness of this tinidazole-containing sequential quadruple therapy compared to placebo on clearing the infection. Subjects were selected from a group of asymptomatic children identified through household visits to 11,365 randomly selected dwelling units. After obtaining parental consent and child assent a total of 1,821 children 3-10 years of age were screened and 235 were positive to a novel urine immunoglobulin class G antibodies test for H. pylori infection and confirmed as infected using a 13C urea breath test, using a hydrolysis urea rate >10 μg/min as cut-off value. Out of those, 119 study subjects had a complete physical exam and baseline blood work and were randomly allocated to four groups, two of which received active H. pylori eradication medication alone or in combination with iron, while the other two received iron only or placebo only. Follow up visits to their houses were done to assess compliance and occurrence of adverse events and at 45+ days post-treatment, a second urea breath test was performed to assess their infection status. The effectiveness was primarily assessed on intent to treat basis (i.e., according to their treatment allocation), and the proportion of those who cleared their infection using a cut-off value >10 μg/min of for urea hydrolysis rate, was the primary outcome. Also we conducted analysis on a per-protocol basis and according to the cytotoxin associated gene A product of the H. pylori infection status. Also we compared the rate of adverse events across the two arms. On intent-to-treat and per-protocol analyses, 44.3% and 52.9%, respectively, of the children receiving the novel quadruple sequential eradication cleared their infection compared to 12.2% and 15.4% in the arms receiving iron or placebo only, respectively. Such differences were statistically significant (p<0.001). The study medications were well accepted and safe. In conclusion, we found in this study population, of mostly asymptomatically H. pylori infected children, living in the US along the border with Mexico, that the quadruple sequential eradication therapy cleared the infection in only half of the children receiving this treatment. Research is needed to assess the antimicrobial susceptibility of the strains of H. pylori infecting this population to formulate more effective therapies. ^

Identifying and Targeting Molecular Deregulations in Uterine Leiomyosarcoma: A Role for mTOR Inhibition-Based Combination Therapies

Uterine leiomyosarcoma (ULMS) is an aggressive malignancy characterized by marked chemoresistance, frequent relapses, and poor outcome. Despite efforts to improve survival over the past several decades, only minimal advances have been made. Hence, there is an urgent and unmet need for better understanding of the molecular deregulations that underlay ULMS and development of more effective therapeutic strategies. This work identified several common deregulations in a large (n=208) tissue microarray of ULMS compared to GI smooth muscle, myometrium, and leiomyoma controls. Our results suggest that significant loss of smooth muscle and gynecological differentiation markers is common in ULMS, a finding that could help render improved ULMS diagnosis, especially for advanced disease. Similarly to reports in other malignancies, we found that several cancer-related proteins were differentially expressed; these could be useful together as biomarkers for ULMS. Notably, we identified significant upregulation and overexpression of the mTOR pathway in ULMS, examined the possible contribution of tyrosine kinase receptor deregulation promoting mTOR activation, and unraveled a role for pS6RP and p4EBP1 as molecular disease prognosticators. The significance of mTOR activation in ULMS and its potential as a therapeutic target were further investigated. Rapamycin abrogated ULMS cell growth and cell cycle progression in vitro but induced only sight growth delay in vivo. Given that effective mTOR therapies likely require combination mTOR blockade with inhibition of other targets, coupled with recent observations suggesting that Aurora A kinase (Aurk A) deregulations commonly occur in ULMS, the preclinical impact of dually targeting both pathways was evaluated. Combined therapy with rapamycin (an mTORC1 inhibitor) and MLN8237 (an investigational Aurk A inhibitor) profoundly and synergistically abrogated ULMS growth in vitro. Interestingly, the superior effects were noted only when MLN8237 was pre-administered. This novel therapeutic combination and scheduling regimen resulted in marked tumor growth inhibition in vivo. Together, these data support further exploration of dual mTOR and Aurk A blockade for the treatment of human ULMS.

Induction of synthetic lethality in mutant KRAS cells for non-small cell lung cancers chemoprevention and therapy

Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Despite improvement in treatment strategies, the 5-year survival rate of lung cancer patients remains low. Thus, effective chemoprevention and treatment approaches are sorely needed. Mutations and activation of KRAS occur frequently in tobacco users and the early stage of development of non-small cell lung cancers (NSCLC). So they are thought to be the primary driver for lung carcinogenesis. My work showed that KRAS mutations and activations modulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors by up-regulating death receptors and down-regulating decoy receptors. In addition, we showed that KRAS suppresses cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of ERK/MAPK-mediated activation of c-MYC which means the mutant KRAS cells could be specifically targeted via TRAIL induced apoptosis. The expression level of Inhibitors of Apoptosis Proteins (IAPs) in mutant KRAS cells is usually high which could be overcome by the second mitochondria-derived activator of caspases (Smac) mimetic. So the combination of TRAIL and Smac mimetic induced the synthetic lethal reaction specifically in the mutant-KRAS cells but not in normal lung cells and wild-type KRAS lung cancer cells. Therefore, a synthetic lethal interaction among TRAIL, Smac mimetic and KRAS mutations could be used as an approach for chemoprevention and treatment of NSCLC with KRAS mutations. Further data in animal experiments showed that short-term, intermittent treatment with TRAIL and Smac mimetic induced apoptosis in mutant KRAS cells and reduced tumor burden in a KRAS-induced pre-malignancy model and mutant KRAS NSCLC xenograft models. These results show the great potential benefit of a selective therapeutic approach for the chemoprevention and treatment of NSCLC with KRAS mutations.

Study on the effect of metformin in patients of metastatic renal cell cancer with diabetes receiving tyrosine kinase inhibitor therapy

Objective: The primary objective of our study was to study the effect of metformin in patients of metastatic renal cell cancer (mRCC) and diabetes who are on treatment with frontline therapy of tyrosine kinase inhibitors. The effect of therapy was described in terms of overall survival and progression free survival. Comparisons were made between group of patients receiving metformin versus group of patients receiving insulin in diabetic patients of metastatic renal cancer on frontline therapy. Exploratory analyses were also done comparing non-diabetic patients of metastatic renal cell cancer receiving frontline therapy compared to diabetic patients of metastatic renal cell cancer receiving metformin therapy. ^ Methods: The study design is a retrospective case series to elaborate the response rate of frontline therapy in combination with metformin for mRCC patients with type 2 diabetes mellitus. The cohort was selected from a database, which was generated for assessing the effect of tyrosine kinase inhibitor therapy associated hypertension in metastatic renal cell cancer at MD Anderson Cancer Center. Patients who had been started on frontline therapy for metastatic renal cell carcinoma from all ethnic and racial backgrounds were selected for the study. The exclusion criteria would be of patients who took frontline therapy for less than 3 months or were lost to follow-up. Our exposure variable was treatment with metformin, which comprised of patients who took metformin for the treatment of type 2 diabetes at any time of diagnosis of metastatic renal cell carcinoma. The outcomes assessed were last available follow-up or date of death for the overall survival and date of progression of disease from their radiological reports for time to progression. The response rates were compared by covariates that are known to be strongly associated with renal cell cancer. ^ Results: For our primary analyses between the insulin and metformin group, there were 82 patients, out of which 50 took insulin therapy and 32 took metformin therapy for type 2 diabetes. For our exploratory analysis, we compared 32 diabetic patients on metformin to 146 non-diabetic patients, not on metformin. Baseline characteristics were compared among the population. The time from the start of treatment until the date of progression of renal cell cancer and date of death or last follow-up were estimated for survival analysis. ^ In our primary analyses, there was a significant difference in the time to progression of patients receiving metformin therapy vs insulin therapy, which was also seen in our exploratory analyses. The median time to progression in primary analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 540 days (95% CI: 350-894) in patients who were receiving insulin therapy (p=0.024). The median time to progression in exploratory analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 279 days (95% CI: 202-372 days) in non-diabetic group (p-value <0.0001). ^ The median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 816 days (95%CI: 558-1405 days) in insulin group (p-value<0.91). For the exploratory analyses, the median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 766 days (95%CI: 649-965 days) in the non-diabetic group (p-value<0.78). Metformin was observed to increase the progression free survival in both the primary and exploratory analyses (HR=0.52 in metformin Vs insulin group and HR=0.36 in metformin Vs non-diabetic group, respectively). ^ Conclusion: In laboratory studies and a few clinical studies metformin has been proven to have dual benefits in patients suffering from cancer and type 2-diabetes via its action on the mammalian target of Rapamycin pathway and effect in decreasing blood sugar by increasing the sensitivity of the insulin receptors to insulin. Several studies in breast cancer patients have documented a beneficial effect (quantified by pathological remission of cancer) of metformin use in patients taking treatment for breast cancer therapy. Combination of metformin therapy in patients taking frontline therapy for renal cell cancer may provide a significant benefit in prolonging the overall survival in patients with metastatic renal cell cancer and diabetes. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

Breast cancer -specific activation of topoisomerase IIalpha and its application in the tumor -targeting gene therapy

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we identified the breast cancer-specific activity of the topoisomerase IIα promoter. We further showed that cdk2 and cyclin A activate topoisomerase IIα promoter in a breast cancer-specific manner. An element containing an inverted CCAAT box (ICB) was shown to respond this signaling. When the ICB-harboring topoisomerase IIα minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent pro-apoptotic gene, was shown to selectively kill breast cancer cells in vitro and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs of CT90-BikDD-treated animals. Finally, we demonstrated that CT90-BikDD treatment potentially enhanced the sensitivity of breast cancer cells to chemotherapeutic agents, especially doxorubicin and taxol. The results indicate that liposomal CT90-BikDD is a novel and effective systemic breast cancer-targeting gene therapy, and its combination with chemotherapy may further improve the current adjuvant therapy for breast cancer. ^

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.