Two-stage stratified designs with survival outcomes and adjustment for misclassification in predictive biomarkers.

Biomarker stratified clinical trial designs are versatile tools to assess biomarker clinical utility and address its relationship with clinical endpoints. Due to imperfect assays and/or classification rules, biomarker status is prone to errors. To account for biomarker misclassification, we consider a two-stage stratified design for survival outcomes with an adjustment for misclassification in predictive biomarkers. Compared to continuous and/or binary outcomes, the test statistics for survival outcomes with an adjustment for biomarker misclassification is much more complicated and needs to take special care. We propose to use the information from the observed biomarker status strata to construct adjusted log-rank statistics for true biomarker status strata. These adjusted log-rank statistics are then used to develop sequential tests for the global (composite) hypothesis and component-wise hypothesis. We discuss the power analysis with the control of the type-I error rate by using the correlations between the adjusted log-rank statistics within and between the design stages. Our method is illustrated with examples of the recent successful development of immunotherapy in nonsmall-cell lung cancer.

Plasma, Prostate and Urine Levels of Tocopherols and Metabolites in Men after Supplementation with a γ-Tocopherol-Rich Vitamin E Mixture.

The vitamin E forms γ- and δ-tocopherols (T) inhibit carcinogenesis in animal models; nevertheless, their cancer preventive activities in humans are uncertain. As an initial step to address this issue, we conducted a pilot phase 0 trial to determine the levels of tocopherols and their metabolites in prostate cancer patients undergoing radical prostatectomy. The patients were randomized to no supplementation or two capsules of a γ-T-rich vitamin E mixture daily for 7 or 14 day prior to prostatectomy. Blood and urine samples were collected before supplementation and on the day of surgery, along with prostate tissue, for analysis of tocopherols and their metabolites. Estimated blood loss during surgery was not significantly different across treatment arms and there were no reported adverse events. Prostate tissue levels of γ-T and δ-T were increased after 14 day of supplementation. Their side-chain degradation metabolites (CEHCs and CMBHCs) were significantly elevated in plasma, prostate and urine samples after supplementation for 7 or 14 day. In conclusion, supplementation with γ-T-rich vitamin E increased the prostate levels of γ-T and δ-T. The use of pure γ-T, δ-T or tocopherol mixtures with higher ratio of γ-T or δ-T to α-T is recommended for future studies.

Data Monitoring for the Chinese Clinical Trials of Remdesivir in Treating Patients with COVID-19 During the Pandemic Crisis.

Two phase-III, double-blind, randomized clinical trials of remdesivir plus SOC (standard of care) versus placebo plus SOC have been conducted in Wuhan hospitals by Chinese investigators during the urgent COVID-19 epidemic [ClincalTrials.gov NCT04257656 and NCT04252664]. These trials have been highly anticipated worldwide. We expect investigators of the trials will soon report the clinical and laboratory findings from the medical perspective. This manuscript provides documentary style information on the process of monitoring key data and making recommendations to the sponsor and investigators based on analytical insights when dealing with the emergent situation from the statistical viewpoint. Having monitored data sequentially from 237 patients, we comment on the strength and weakness of the study design and suggest the treatment effect of remdesivir on severe COVID-19 cases. Our experience with using the Dynamic Data Monitoring (DDM) tool has demonstrated its efficiency and reliability in supporting DSMB's instantaneous review of essential data during the emergent situation. DDM, when used properly by disciplined statisticians, has shown its capability of exploring the trial data flexibly and, in the meantime, protecting the trial's scientific integrity.

Two-stage enrichment clinical trial design with adjustment for misclassification in predictive biomarkers.

A two-stage enrichment design is a type of adaptive design, which extends a stratified design with a futility analysis on the marker negative cohort at the first stage, and the second stage can be either a targeted design with only the marker positive stratum, or still the stratified design with both marker strata, depending on the result of the interim futility analysis. In this paper, we consider the situation where the marker assay and the classification rule are possibly subject to error. We derive the sequential tests for the global hypothesis as well as the component tests for the overall cohort and the marker-positive cohort. We discuss the power analysis with the control of the type I error rate and show the adverse impact of the misclassification on the powers. We also show the enhanced power of the two-stage enrichment over the one-stage design and illustrate with examples of the recent successful development of immunotherapy in non-small-cell lung cancer.

Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.

A phase II trial of riluzole, an antagonist of metabotropic glutamate receptor 1 (GRM1) signaling, in patients with advanced melanoma.

Studies demonstrate that GRM, expressed by >60% of human melanomas, may be a therapeutic target. We performed a phase II trial of 100 mg PO bid of riluzole, an inhibitor of GRM1 signaling, in patients with advanced melanoma with the primary endpoint of response rate. Thirteen patients with GRM1-positive tumors were enrolled. No objective responses were observed, and accrual was stopped. Stable disease was noted in six (46%) patients, with one patient on study for 42 weeks. Riluzole was well tolerated, with fatigue (62%) as the most common adverse event. Downregulation of MAPK and PI3K/AKT was noted in 33% of paired tumor biopsies. Hypothesis-generating correlative studies suggested that downregulation of angiogenic markers and increased leukocytes at the active edge of tumor correlate with clinical benefit. Pharmacokinetic analysis showed interpatient variability consistent with prior riluzole studies. Future investigations should interrogate mechanisms of biologic activity and advance the development of agents with improved bioavailability.

Phase 2 trial of paclitaxel, 13-cis retinoic acid, and interferon alfa-2b in the treatment of advanced stage or recurrent cervical cancer.

OBJECTIVE: Overexpression of bcl-2 is a mechanism of drug resistance in cervical cancer. Agents that down-regulate bcl-2 may decrease tumor cell threshold and sensitize tumor cells to chemotherapy. The objective of this multi-institutional phase 2 trial was to evaluate the efficacy and toxicity of paclitaxel and bcl-2 modulators (13-cis retinoic acid and interferon alfa-2b) in patients with advanced-stage or recurrent cervical cancer. MATERIALS AND METHODS: Patients had biopsy-proven metastatic, first relapse, or persistent cervical cancer with no prior chemotherapy except for chemosensitizing agents. The treatment consisted of oral 13-cis retinoic acid, 1 mg/kg, and subcutaneous interferon alfa-2b, 6 mU/m, days 1 to 4, and intravenous paclitaxel, 175 mg/m, day 4 until disease progression or adverse events prohibited treatment. The primary endpoint was overall response rate. RESULTS: Thirty-three patients were enrolled between March 2001 and June 2009. Thirty-one patients were eligible for evaluation of treatment response. Twenty-seven patients (82%) received prior concurrent chemoradiation or radiotherapy alone before study enrollment. The overall response rate was 30% (6 complete responses and 4 partial responses). Furthermore, 7 patients (21%) had stable disease. Grade 3 or 4 adverse events included neutropenia (n =16 [48%]), febrile neutropenia (n = 1 [3%]), and anemia (n = 1 [3%]). There were no treatment-related deaths. The median progression-free survival was 3.4 months (95% confidence interval, 2.0-7.4 months), and overall survival was 11.2 months (95% confidence interval, 7.5-26.2 months). Of 6 patients with complete responses, 5 patients survived more than 2 years. CONCLUSIONS: Combination therapy with paclitaxel, 13-cis retinoic acid, and interferon alfa-2b is feasible and safe in treating patients with advanced and recurrent cervical cancer.

Methods of designing two-stage winner trials with survival outcomes.

In drug development, especially for oncology studies, a recent proposal is to combine a costly phase II dose selection study with a subsequent phase III study into a single trial that compares the selected (winning) dose from the first stage with the control group. This design may also be used in phase III trials, in which the winning active treatment regimen, selected at the first stage, is compared with the control group at the second stage. This design is known as a two-stage winner design, as proposed by Shun et al. (2008) for continuous outcomes. Time-to-event data are often analyzed in oncology trials. In order to derive the critical value and power of this design, per Shun et al. (2008), it is essential to calculate the asymptotic covariance and correlation of the log-rank statistics for survival outcomes between the two stages. In this paper, we derive the asymptotic covariance and correlation, and provide additional approximate design parameters. Examples are given to illustrate the method, and simulations are performed to evaluate the veracity of these approximate design parameters.

δ-tocopherol is more active than α - or γ -tocopherol in inhibiting lung tumorigenesis in vivo.

In contrast to strong epidemiologic, preclinical, and secondary clinical evidence for vitamin E (tocopherols) in reducing cancer risk, large-scale clinical cancer-prevention trials of α-tocopherol have been negative. This vexing contrast helped spur substantial preclinical efforts to better understand and improve the antineoplastic activity of tocopherol through, for example, the study of different tocopherol forms. We previously showed that the γ-tocopherol-rich mixture (γ-TmT) effectively inhibited colon and lung carcinogenesis and the growth of transplanted lung-cancer cells in mice. We designed this study to determine the relative activities of different forms of tocopherol in a xenograft model, comparing the anticancer activities of δ-tocopherol with those of α- and γ-tocopherols. We subcutaneously injected human lung cancer H1299 cells into NCr nu/nu mice, which then received α-, γ-, or δ-tocopherol or γ-TmT in the diet (each at 0.17% and 0.3%) for 49 days. δ-Tocopherol inhibited tumor growth most strongly. γ-Tocopherol and γ-TmT (at 0.3%) also inhibited growth significantly, but α-tocopherol did not. δ-Tocopherol also effectively decreased oxidative DNA damage and nitrotyrosine formation and enhanced apoptosis in tumor cells; again, γ-tocopherol also was active in these regards but less so, and α-tocopherol was not. Each supplemented diet increased serum levels of its tocopherol - up to 45 µmol/L for α-tocopherol, 9.7 µmol/L for γ-tocopherol, and 1.2 µmol/L for δ-tocopherol; dietary γ- or δ-tocopherol, however, decreased serum α-tocopherol levels, and dietary α-tocopherol decreased serum levels of γ-tocopherol. Each dietary tocopherol also increased its corresponding side-chain-degradation metabolites, with concentrations of δ-tocopherol metabolites greater than γ-tocopherol and far greater than α-tocopherol metabolites in serum and tumors. This study is the first in vivo assessment of δ-tocopherol in tumorigenesis and shows that δ-tocopherol is more active than α- or γ-tocopherol in inhibiting tumor growth, possibly through trapping reactive oxygen and nitrogen species and inducing apoptosis; δ-tocopherol metabolites could contribute significantly to these results.

A phase 0 trial of riluzole in patients with resectable stage III and IV melanoma.

PURPOSE: Ectopic expression of GRM1 in murine melanocytes results in transformation into a form of melanoma, and more than 60% of human melanoma samples tested ectopically express GRM1. Stimulation of this receptor in vitro results in up-regulation of activated extracellular signal-regulated kinase (ERK). Furthermore, a xenograft model of melanoma treated with riluzole, an oral GRM1 blocking agent, showed decreased tumor growth compared with the untreated controls. We have now completed a phase 0 trial of riluzole in patients with melanoma. EXPERIMENTAL DESIGN: Patients enrolled on this trial underwent a pretreatment biopsy, took 200 mg of oral riluzole per day for 14 days, and then underwent resection of their remaining tumor. We compared the levels of pERK and pAKT in the pretreatment and post-treatment samples and assessed the metabolic activity of pretreatment and post-treatment tumors using fluorodeoxyglucose positron emission tomography (FDG-PET) scanning. RESULTS: We accrued 12 patients and all expressed GRM1. We found a significant decrease in pAKT and/or pERK in post-treatment tumor samples as compared with pretreatment samples in 4 (34%) patients. These four patients had a significant decrease in FDG-PET intensity post-treatment as well. Two other patients had a clinical response with no corresponding metabolic response; five patients had similar pretreatment and post-treatment FDG-PET scan findings; and one patient had progressive disease. CONCLUSIONS: Our data show that glutamate blockade with riluzole can inhibit signaling through the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT pathways and suppress the metabolic activity of melanoma. The ectopic expression of metabotropic glutamate receptors may be important in the pathogenesis of human melanoma, and targeting this pathway may be an effective therapy.

Tumorigenic effect of some commonly used moisturizing creams when applied topically to UVB-pretreated high-risk mice.

Irradiation of SKH-1 mice with UVB (30 mJ cm(-2)) twice a week for 20 weeks resulted in mice with a high risk of developing skin tumors over the next several months in the absence of further irradiation with UVB (high-risk mice). Topical applications of 100 mg of Dermabase, Dermovan, Eucerin Original Moisturizing Cream (Eucerin), or Vanicream once a day, 5 days a week for 17 weeks to these high-risk mice increased significantly the rate of formation of tumors and the rate of increase in tumor size per mouse. Additional studies indicated that treatment of high-risk mice with Dermabase, Dermovan, Eucerin, or Vanicream for 17 weeks increased the total number of histologically characterized tumors by 69% (average of two experiments; P<0.0001 in each experiment), 95% (P<0.0001), 24% (P<0.01), and 58% (P<0.0001), respectively. Topical applications of a specially designed Custom Blend cream to high-risk mice was not tumorigenic. The results indicate that several commercially available moisturizing creams increase the rate of formation and number of tumors when applied topically to UVB-pretreated high-risk mice. Further studies are needed to determine the effects of topical applications of moisturizing creams on sunlight-induced skin cancer in humans.

Approaches to handling data when a phase II trial deviates from the pre-specified Simon's two-stage design.

Simon's 'optimal' and 'minimax' two-stage designs are common methods for conducting phase IIA studies investigating new cancer therapies. However, these designs are rather rigid in their settings because of the pre-specified rejection rules and fixed sample sizes at each stage. In practice, we often encounter the problem that a study is unable to adhere to the event number and sample sizes of the original two-stage design. In this paper, we consider some approaches in handling situations where deviations or interruptions from the original Simon's two-stage design occur because recruitment of patients is slower than expected. We consider four scenarios and use conditional probabilities to address the issues commonly inquired by the scientific review board. We also discuss how to report p-values in these situations.

Elicitation of a beta prior for Bayesian inference in clinical trials.

When making Bayesian inferences we need to elicit an expert's opinion to set up the prior distribution. For applications in clinical trials, we study this problem with binary variables. A critical and often ignored issue in the process of eliciting priors in clinical trials is that medical investigators can seldom specify the prior quantities with precision. In this paper, we discuss several methods of eliciting beta priors from clinical information, and we use simulations to conduct sensitivity analyses of the effect of imprecise assessment of the prior information. These results provide useful guidance for choosing methods of eliciting the prior information in practice.

Clinical and biological activity of soy protein powder supplementation in healthy male volunteers.

PURPOSE: To determine if a commonly used soy protein supplement exhibits biological activity in vivo and in vitro, we evaluated an over-the-counter soy protein powder supplement using blood from healthy male volunteers and in an estrogen receptor in vitro assay. SUBJECTS AND METHODS: We recruited healthy male volunteers 18 years of age or older that were in good health. Treatment consisted of consuming two scoops (56 g) of pure soy protein powder (Puritan's Pride, Oakdale, NY) daily for 28 days. Serum testosterone and luteinizing hormone (LH) levels were collected on days -7, 0, 14, and 28 of therapy, and day 42. A reporter estrogen receptor (ER) assay was used to determine the effect on ER-beta and ER-alpha in vitro. RESULTS: Twelve subjects were enrolled with a mean age of 32.25 years (range 25 to 47). Serum testosterone decreased 19%(+/-22%) during the 4-week use of soy protein powder (P = 0.021) and increased within 2 weeks after we discontinued soy protein powder. Serum LH concentrations decreased during the 4-week use of soy protein powder then increased within 2 weeks after we stopped the soy protein powder, but the changes did not reach statistical significance (P = 0.20). Soy protein powder was found to induce agonist activity to ER-beta using a reporter estrogen receptor assay in yeast. CONCLUSION: Soy protein powder decreases serum testosterone levels in healthy men and acts as an ER-beta agonist; the significance of this biological effect with respect to cancer prevention needs further study.

The CD44 receptor interacts with P-glycoprotein to promote cell migration and invasion in cancer.

Invasion and metastases of cancer cells and the development of resistance to anticancer therapies are the main causes of morbidity and mortality from cancer. For more than two decades, these two important but not clearly related aspects in the biology of cancer have been extensively studied. Specifically, P-glycoprotein and CD44 have been characterized and are known to be determinants of multidrug resistance (MDR) and metastases. Despite this body of knowledge, few reports have linked the two phenotypes and only recently have there been reasons to suspect a direct connection. In this report, we show that a novel physical and genetic interaction between CD44s and P-glycoprotein is in part responsible for the correlation between MDR and invasive potential in cancer cells. P-glycoprotein-specific substrates that interfere with its function reduced in vitro invasion, migration, and the physical colocalization of CD44s and P-glycoprotein. CD44 expression in sensitive cells promoted the expression of P-glycoprotein and the MDR phenotype. RNA interference of MDR1 inhibited the rate of cell migration. These data indicate that there is a close interaction between CD44 and P-glycoprotein that results in the concurrent expression and modulation of two malignant phenotypes, invasion and MDR.

Effect of docetaxel in patients with hormone-dependent prostate-specific antigen progression after local therapy for prostate cancer.

PURPOSE: To evaluate docetaxel in the treatment of patients with early-stage prostate cancer with prostate-specific antigen (PSA) progression after local therapy without androgen ablation therapy. PATIENTS AND METHODS: Twenty-five patients with adenocarcinoma of the prostate with PSA progression despite local therapy were treated with 70 mg/m2 docetaxel every 21 days. Treatment was planned for eight cycles. Patients were followed up for effects on PSA, testosterone, and toxicity. RESULTS: Twenty-three of 25 patients completed at least one full cycle of therapy. Ten (43%) of 23 patients demonstrated a decrease in PSA by > or = 50% for at least 4 weeks. The nadir decrease in PSA occurred beyond 150 days of therapy in most patients. Therapy was well tolerated. Grade 4 neutropenia with fever occurred in only six cycles (4.5%). Two patients required 25% dose reductions, both occurring with cycle 6, secondary to increased transaminases in one patient, and grade 3 lacrimation in the other patient. Two patients were removed after the first cycle of therapy due to toxicity (deep venous thrombosis, chest palpitations). Mean testosterone levels were not reduced in 17 patients assessed before and during therapy (P = .12). CONCLUSION: This study demonstrated the activity of docetaxel alone, without androgen ablation, in patients with PSA progression after completion of local therapy. Treatment with docetaxel in this population with early disease progression was well tolerated, biochemically active, and was not androgen ablative. Accrual to national phase III studies in early disease is now critical and should be strongly encouraged to determine the ability of early chemotherapy to improve survival.

A phase II trial of imatinib mesylate in patients with prostate specific antigen progression after local therapy for prostate cancer.

PURPOSE: To test the hypothesis that progression of androgen sensitive prostate cancer is dependent on growth factors, such as platelet derived growth factor (PDGF), and inhibition of PDGF receptor (PDGF-R) with imatinib will induce anti-tumor activity. PATIENTS AND METHODS: This phase II study evaluated imatinib in patients with androgen sensitive prostate cancer and prostate specific antigen (PSA) progression after local therapy. Patients received 400 mg of imatinib orally twice a day for 24 weeks (six cycles). Patients were monitored every 4 weeks for an effect on PSA and toxicity. Immunohistochemistry (IHC) for PDGF-R was performed in available tumor specimens. RESULTS: Twenty-one patients were enrolled on this trial with a median age of 64 years. A total of 72 cycles of therapy were administered. Sixteen patients were evaluable for a response. Nine of the 16 patients demonstrated a stable PSA. Seven patients demonstrated PSA progression. Grade 3 and 4 toxicity included rash (4.1%), hematuria (1.4%), diarrhea (1.4%), and neutropenia (2.7%). Testosterone levels did not change during therapy. Four patients with available tumor demonstrated PDGF-R alpha and beta by IHC. CONCLUSIONS: This first study evaluated the efficacy and safety of imatinib in patients with early androgen sensitive prostate cancer following local therapy. As a single agent at this dosing, imatinib had limited biochemical activity.

Two-stage adaptive strategy for superiority and non-inferiority hypotheses in active controlled clinical trials.

In active controlled trials without a placebo arm, there are usually two study objectives: to test a superiority hypothesis that the experimental treatment is more effective than the active control therapy, and to test a non-inferiority hypothesis that the experimental treatment is therapeutically no worse than the active control within a defined margin. For a two-stage adaptive design, it is not necessary to give a fixed sample size calculation at the planning stage of the study when treatment effect information is often insufficient. Instead, decision and estimation of the design specifications can be made more reliably after the first stage when interim results are available. We propose the use of conditional power approach to determine the sample size and critical values for testing the superiority and non-inferiority hypotheses for the second stage based on the observed result of the first stage. The proposed adaptive procedure preserves the overall type I error rate for both superiority and non-inferiority, and has the flexibility of early termination of the study (for futility or efficacy) or extending the study by appropriate sample size.

Adaptive two-stage designs for single-arm phase IIA cancer clinical trials.

The main purpose of a phase IIA trial of a new anticancer therapy is to determine whether the therapy has sufficient promise against a specific type of tumor to warrant its further development. The therapy will be rejected for further investigation if the true response rate is less than some uninteresting level and the test of hypothesis is powered at a specific target response rate. Two-stage designs are commonly used for this situation. However, in many situations investigators often express concern about uncertainty in targeting the alternative hypothesis to study power at the planning stage. In this article, motivated by a real example, we propose a strategy for adaptive two-stage designs that will use the information at the first stage of the study to either reject the therapy or continue testing with either an optimistic or a skeptic target response rate, while the type I error rate is controlled. We also introduce new optimal criteria to reduce the expected total sample size.

Development of a bioanalytical liquid chromatography method for quantitation of 9-nitrocamptothecin in human plasma.

9-Nitrocamptothecin (9-NC) is an orally administered camptothecin (CPT) that is under evaluation in clinical trials. This compound is not fluorescent, which has hampered development of a sensitive high-performance liquid chromatographic (LC) assay for measurement of drug concentrations in clinical trials. We now report development of an assay that involves reduction of 9-NC to the fluorescent compound 9-aminocamptothecin (9-AC). The method is based on enzymatic reduction of 9-NC using bovine liver S-9 fraction. This method is validated to quantitate 9-NC and 9-AC in patient samples, and yields results comparable to those obtained with an LC/MS method.