Monitoring futility and efficacy in phase II trials with Bayesian posterior distributions-A calibration approach.

A multistage single arm phase II trial with binary endpoint is considered. Bayesian posterior probabilities are used to monitor futility in interim analyses and efficacy in the final analysis. For a beta-binomial model, decision rules based on Bayesian posterior probabilities are converted to "traditional" decision rules in terms of number of responders among patients observed so far. Analytical derivations are given for the probability of stopping for futility and for the probability to declare efficacy. A workflow is presented on how to select the parameters specifying the Bayesian design, and the operating characteristics of the design are investigated. It is outlined how the presented approach can be transferred to statistical models other than the beta-binomial model.

Early metabolic response in sequential FDG-PET/CT under cetuximab is a predictive marker for clinical response in first-line metastatic colorectal cancer patients: results of the phase II REMOTUX trial.

BACKGROUND: To assess the predictive value of early metabolic response (ΔSUV) after short-term treatment with first-line cetuximab in patients (pts) with RAS-wt metastatic colorectal cancer (mCRC). METHODS: In this prospective phase II study, RAS-wt mCRC pts received a single-agent cetuximab run-in therapy of 2 weeks. ΔSUV was assessed with FDG-PET/CT on days 0 and 14. Early clinical response (ECR) was evaluated with CT on day 56 after treatment with FOLFIRI-cetuximab. Primary endpoint was the predictive significance of ΔSUV for ECR. Secondary endpoints were PFS (progression free survival), OS and the influence of ΔSUV on survival. RESULTS: Forty pts were enroled and 33 pts were evaluable for the primary endpoint. The CT response rate was 57.6%. For responders, ΔSUV was significantly higher (p = 0.0092). A significant association of ΔSUV with ECR was found (p = 0.02). Median PFS was 11.7 months and median OS was 33.5 months with a 1-year survival rate of 87.9%. ΔSUV was found to significantly impact the hazard for OS (p = 0.045). CONCLUSIONS: We demonstrate that cetuximab induces metabolic responses in mCRC pts. The study endpoint was met with the ΔSUV discriminating between responders and non-responders. However, these data should be validated in larger patient cohorts.

POSITIVE study: physical exercise program in non-operable lung cancer patients undergoing palliative treatment.

BACKGROUND: Patients with advanced stage non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) often experience multidimensional impairments, affecting quality of life during their course of disease. In lung cancer patients with operable disease, several studies have shown that exercise has a positive impact on quality of life and physical functioning. There is limited evidence regarding efficacy for advanced lung cancer patients undergoing palliative treatment. Therefore, the POSITIVE study aims to evaluate the benefit of a 24-week exercise intervention during palliative treatment in a randomized controlled setting. METHODS/DESIGN: The POSITIVE study is a randomized, controlled trial investigating the effects of a 24-week exercise intervention during palliative treatment on quality of life, physical performance and immune function in advanced, non-operable lung cancer patients. 250 patients will be recruited in the Clinic for Thoracic Diseases in Heidelberg, enrolment begun in November 2013. Main inclusion criterion is histologically confirmed NSCLC (stage IIIa, IIIb, IV) or SCLC (Limited Disease-SCLC, Extensive Disease-SCLC) not amenable to surgery. Patients are randomized into two groups. Both groups receive weekly care management phone calls (CMPCs) with the goal to assess symptoms and side effects. Additionally, one group receives a combined resistance and endurance training (3x/week). Primary endpoints are quality of life assessed by the Functional Assessment of Cancer Therapy for patients with lung cancer (FACT-L, subcategory Physical Well-Being) and General Fatigue measured by the Multidimensional Fatigue Inventory (MFI-20). Secondary endpoints are physical performance (maximal voluntary isometric contraction, 6-min walk distance), psychosocial (depression and anxiety) and immunological parameters and overall survival. DISCUSSION: The aim of the POSITIVE trial is the evaluation of effects of a 24-week structured and guided exercise intervention during palliative treatment stages. Analysis of various outcomes (such as quality of life, physical performance, self-efficacy, psychosocial and immunological parameters) will contribute to a better understanding of the potential of exercise in advanced lung cancer patients. In contrast to other studies with advanced oncological patients the POSITIVE trial provides weekly phone calls to support patients both in the intervention and control group and to segregate the impact of physical activity on quality of life. TRIAL REGISTRATION: ClinicalTrials.gov NCT02055508 (Date: December 12, 2013).

Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides and their evaluation as ligands for NMDA receptor glycine binding site.

A series of 2-substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides were synthesized and evaluated for their affinity to the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor. The binding affinity was determined by the displacement of radioligand [(3)H]MDL-105,519 from rat cortical membrane preparations. The most attractive structures in the search for prospective NMDA receptor ligands were identified to be 2-arylcarbonylmethyl substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides. It has been demonstrated for the first time that the replacement of NH group in the ligand by sp(3) CH2 is tolerated. This finding may pave the way for previously unexplored approaches for designing new ligands of the NMDA receptor.

Some Issues of Sample Size Calculation for Time-to-Event Endpoints Using the Freedman and Schoenfeld Formulas.

This article deals with seven special issues related to the assumptions, applicability, and practical use of formulas for calculating power or sample size, respectively, for comparative clinical trials with time-to-event endpoints, with particular focus on the well-known Freedman and Schoenfeld methods. All problems addressed are illustrated by numerical examples, and recommendations are given on how to deal with them in the planning of clinical trials.

Chemotherapy for advanced pancreatic adenocarcinoma in elderly patients (≥70 years of age): a retrospective cohort study at the National Center for Tumor Diseases Heidelberg.

BACKGROUND: Pancreatic cancer is mainly a disease of the elderly population, but clinical trials do not reflect this age distribution. Data on treatment strategies and outcome of older patients are limited. The aim of our study was to analyze safety and outcome in elderly patients with advanced pancreatic cancer treated with palliative chemotherapy at the outpatient clinic of the National Center for Tumor Diseases (NCT) at Heidelberg University Hospital. MATERIALS AND METHODS: We retrospectively analyzed 53 patients ≥70 years using a prospectively maintained database. Requirements were (1) histologically proven diagnosis of ductal pancreatic adenocarcinoma, (2) age ≥70 at time of diagnosis of advanced disease, and (3) measurable advanced disease. RESULTS: The median age was 73 years. 81% of the patients received a gemcitabine-based first-line therapy. Median overall survival was 6.7 months. Survival differed significantly between patient groups with low (≤1) and high (≥2) Eastern Cooperative Oncology Group performance status (7.8 vs. 3.9 months, p = 0.002). 30.2% of the patients developed side effects resulting in dosage reductions. 39.6% of the patients received second-line treatment. Residual survival after disease progression was significantly longer for second-line treatment compared to best supportive care (151 vs. 39 days, p = 0.019). CONCLUSIONS: Overall, our older patients did not have an inferior outcome compared to the reported trial populations that included younger patients. Thus, palliative chemotherapy should be considered independently from chronological age, but the performance status should be carefully noticed. Second-line therapy should be considered for patients in good performance status after first progression.

Scaffold hopping approach towards various AFQ-056 analogs as potent metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate receptor subtype 5 has evolved into a promising target for the treatment of various diseases of the central nervous system, such as Fragile X and L-DOPA induced dyskinesia. One of the most advanced clinical compound is Novartis' AFQ-056 (Mavoglurant), which served us as a template for a scaffold hopping approach, generating a structurally diverse set of potent analogs. Both the limited aqueous solubility and the relatively poor metabolic stability of AFQ-056 were improved with hexahydrocyclopenta[c]pyrrole derivative 54a, which proved to be a valuable candidate for further development.

Discovery, synthesis, and structure-activity relationships of 2-aminoquinazoline derivatives as a novel class of metabotropic glutamate receptor 5 negative allosteric modulators.

A virtual screening approach using various in silico methodologies led to the discovery of 2-(m-tolylamino)-7,8-dihydroquinazolin-5(6H)-one (1) as a moderately active negative allosteric modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) showing high selectivity against the subtype mGluR1. Modifications of the parent compound by rational design yielded a series of highly potent derivatives which will serve as valuable starting points for further hit-to-lead optimization efforts toward a suitable drug candidate for the treatment of L-DOPA induced dyskinesia.

Investigation of the GnRH antagonist degarelix isomerization in biological matrices.

One of the main objectives of peptide drug design is the improvement of peptide pharmacokinetics with maintaining biological activity, which can be achieved by the complex modifications of the primary structure of the peptides. However, these changes often lead to the formation of peculiar impurities in the peptide drugs and their metabolites, which require the development of advanced analytical methods to properly assess their content. Here, we investigated the degradation of the potent long-acting GnRH antagonist degarelix in various biologic media by the tailor-made HPLC method, which allows precise determination of 5-Aph(Hyd)-degarelix isomer, an impurity found in the degarelix active pharmaceutical ingredient (API) during its manufacturing. Unexpectedly, we discovered a rapid and irreversible conversion of degarelix API into the corresponding hydantoin isomer in serum, suggesting that this impurity can be also a potential drug metabolite in vivo. This finding underlines the importance of the development of more accurate and performing analytical techniques to correctly characterize the chemical composition of the manufactured drugs and their behavior under physiological conditions.

A phase II study for metabolic in vivo response monitoring with sequential 18FDG-PET-CT during treatment with the EGFR-monoclonal-antibody cetuximab in metastatic colorectal cancer: the Heidelberg REMOTUX trial.

BACKGROUND: The epidermal growth factor receptor monoclonal antibody cetuximab has proven activity in metastatic colorectal cancer. To date, the mechanisms of action are not completely understood. Especially the impact on tumor glucose metabolism, or tumor vascularization remains largely unclear. The understanding of mechanisms such as early changes in tumor metabolism is of clinical importance since there may be a substantial influence on choice and sequence of drug combinations. Early signals of response to cetuximab may prove useful to identify patients having a relevant clinical treatment benefit. The objective of this trial is to evaluate the predictive relevance of the relative change in (18)F-Fluorodeoxyglucose tumor uptake for early clinical response during short-term single agent treatment with cetuximab. Early clinical response will be routinely measured according to the response evaluation criteria in solid tumors. Accompanying research includes cytokine immune monitoring and analysis of tumor proteins and tumor genes. METHODS/DESIGN: The REMOTUX trial is an investigator-initiated, prospective, open-label, single-arm, single-center early exploratory predictive study. The first (18)F-FDG PET-CT is conducted at baseline followed by the run-in phase with cetuximab at days 1 and 8. At day 14, the second (18)F-FDG PET-CT is performed. Subsequently, patients are treated according to the Folfiri-cetuximab regimen as an active and approved first-line regimen for metastatic colorectal carcinoma. At day 56, clinical response is evaluated with a CT-scan compared to the baseline analysis. Tracer uptake is assessed using standardized uptake values (SUVs). The main hypothesis to be tested in the primary analysis is whether or not the relative change in the SUV from baseline to day 14 has any predictive relevance for early clinical response determined at day 56. Patients are followed until death from any cause or until 24 months after the last patient has ended trial treatment. DISCUSSION: The aim of this trial is to evaluate metabolic changes in metastatic colorectal cancer during short-term single agent treatment with cetuximab and to analyse their potential of predicting early clinical response. This could be helpful to answer the question if early identification of patients not responding to cetuximab is possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT200811021020; EudraCT 200901327923.

Lentiviral vector integration profiles differ in rodent postmitotic tissues.

Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.

Insertion sites in engrafted cells cluster within a limited repertoire of genomic areas after gammaretroviral vector gene therapy.

Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.

Sequential FDG-PET and induction chemotherapy in locally advanced adenocarcinoma of the Oesophago-gastric junction (AEG): the Heidelberg Imaging program in Cancer of the oesophago-gastric junction during Neoadjuvant treatment: HICON trial.

BACKGROUND: 18-Fluorodeoxyglucose-PET (18F-FDG-PET) can be used for early response assessment in patients with locally advanced adenocarcinomas of the oesophagogastric junction (AEG) undergoing neoadjuvant chemotherapy. It has been recently shown in the MUNICON trials that response-guided treatment algorithms based on early changes of the FDG tumor uptake detected by PET are feasible and that they can be implemented into clinical practice. Only 40%-50% of the patients respond metabolically to therapy. As metabolic non-response is known to be associated with a dismal prognosis, metabolic non-responders are increasingly treated with alternative neoadjuvant chemotherapies or chemoradiation in order to improve their clinical outcome. We plan to investigate whether PET can be used as response assessment during radiochemotherapy given as salvage treatment in early metabolic non-responders to standard chemotherapy. METHODS/DESIGN: The HICON trial is a prospective, non-randomized, explorative imaging study evaluating the value of PET as a predictor of histopathological response in metabolic non-responders. Patients with resectable AEG type I and II according to Siewerts classification, staged cT3/4 and/or cN+ and cM0 by endoscopic ultrasound, spiral CT or MRI and FDG-PET are eligible. Tumors must be potentially R0 resectable and must have a sufficient FDG-baseline uptake. Only metabolic non-responders, showing a < 35% decrease of SUV two weeks after the start of neoadjuvant chemotherapy are eligible for the study and are taken to intensified taxane-based RCT (chemoradiotherapy (45 Gy) before surgery. 18FDG-PET scans will be performed before ( = Baseline) and after 14 days of standard neoadjuvant therapy as well as after the first cycle of salvage docetaxel/cisplatin chemotherapy (PET 1) and at the end of radiochemotherapy (PET2). Tracer uptake will be assessed semiquantitatively using standardized uptake values (SUV). The percentage difference ΔSUV = 100 (SUV Baseline - SUV PET1)/SUV Baseline will be calculated and assessed as an early predictor of histopathological response. In a secondary analysis, the association between the difference SUV PET1 - SUV PET2 and histopathological response will be evaluated. DISCUSSION: The aim of this study is to investigate the potential of sequential 18FDG-PET in predicting histopathological response in AEG tumors to salvage neoadjuvant radiochemotherapy in patients who do not show metabolic response to standard neoadjuvant chemotherapy.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Synthesis and pharmacological validation of a novel series of non-steroidal FXR agonists.

To overcome the known liabilities of GW4064 a series of analogs were synthesized where the stilbene double bond is replaced by an oxymethylene or amino-methylene linker connecting a terminal benzoic acid with a substituted heteroaryl in the middle ring position. As a result we discovered compounds with increased potency in vitro that cause dose-dependent reduction of plasma triglycerides and cholesterol in db/db mice down to 2 x 1 mg/kg/day upon oral administration.

A randomized multicentre phase II trial comparing adjuvant therapy in patients with interferon alpha-2b and 5-FU alone or in combination with either external radiation treatment and cisplatin (CapRI) or radiation alone regarding event-free survival - CapRI-2.

BACKGROUND: The 5-year survival of patients with resected pancreatic adenocarcinoma is still unsatisfying. The ESPAC-1 and the CONKO 001 trial proofed that adjuvant chemotherapy improves 5-year survival significantly from approximately 14% to 21%. In parallel, investigators from the Virginia Mason Clinic reported a 5-year survival rate of 55% in a phase II trial evaluating a combination of adjuvant chemotherapy, immunotherapy and external beam radiation (CapRI-scheme). Two other groups confirmed in phase II trials these results to a certain extent. However, these groups reported severe gastrointestinal toxicity (up to 93% grade 3 or 4 toxicity). In a randomized controlled phase III trial, called CapRI, 110 patients were enrolled from 2004 to 2007 in Germany and Italy to check for reproducibility. Interestingly, much less gastrointestinal toxicity was observed. However, dose-reduction due to haematological side effects had to be performed in nearly all patients. First clinical results are expected for the end of 2009. METHODS/DESIGN: CapRI-2 is an open, controlled, prospective, randomized, multicentre phase II trial with three parallel arms. A de-escalation of the CapRI-scheme will be tested in two different modifications. Patients in study arm A will be treated as outpatients with the complete CapRI-scheme consisting of cisplatin, Interferon alpha-2b and external beam radiation and three cycles of 5-fluorouracil continuous infusion. In study arm B the first de-escalation will be realised by omitting cisplatin. Next, patients in study arm C will additionally not receive external beam radiation. A total of 135 patients with pathologically confirmed R0 or R1 resected pancreatic adenocarcinoma are planned to be enrolled. Primary endpoint is the comparison of the treatment groups with respect to six-month event-free-survival. An event is defined as grade 3 or grade 4 toxicity, objective tumour recurrence, or death. DISCUSSION: The aim of this clinical trial is to evaluate de-escalation of the CapRI-scheme. It is hypothesised that removal of cisplatin and radiotherapy will have no significant effect or only a minor impact on the clinical response but result in substantially lower toxicity. TRIAL REGISTRATION: Current Controlled Trials ISRCTN79802092.

Short time to progression under first-line chemotherapy is a negative prognostic factor for time to progression and residual survival under second-line chemotherapy in advanced pancreatic cancer.

OBJECTIVES: Second-line chemotherapy is widely used in advanced pancreatic cancer. However, only few data exist concerning the question who might benefit from second-line therapy. We intended to identify prognostic factors for time to second progression (TTP2) and residual survival following second-line chemotherapy of patients with advanced pancreatic cancer. METHODS: We performed a retrospective cohort study on 78 patients who started palliative chemotherapy at our department from January 2004 to June 2006 due to advanced adenocarcinoma of the pancreas. 46 patients who progressed following first-line therapy were analyzed. RESULTS: In multivariate analyses, time to first progression (TTP1) <6 months and elevated LDH at the time of diagnosis were shown to be strong and highly significant independent prognostic factors for TTP2 and residual survival. For patients with TTP1 <6 months, prognosis was poor with a median residual survival of 4.4 versus 7.5 months for those with TTP1 > or =6 months. CONCLUSION: In our cohort of patients with advanced pancreatic cancer treated with second-line chemotherapy, TTP1 <6 months is a strong negative prognostic factor for TTP2 and residual survival.

Safety of the use of Tissucol Duo S in cardiovascular surgery: retrospective analysis of 2149 patients after coronary artery bypass grafting.

OBJECTIVE: Fibrin sealant is widely used in almost all fields of surgery and has proved to be an effective therapeutic tool in cardiothoracic surgery. Nevertheless, there have been concerns about early bypass graft occlusion associated with the use of fibrin glue. This analysis has been performed to assess the risks and benefits of Tissucol Duo S in coronary artery bypass grafting (CABG) surgery. METHODS: Two thousand one hundred forty-nine patients were included in this retrospective study, 879 (40.9%) were intra-operatively treated with Tissucol Duo S fibrin glue, 1270 (59.1%) did not receive fibrin glue (control group). Patient characteristics were documented according to the EuroScore. Intra- and postoperative data were collected. Primary endpoint of this study was the 30-day all-cause mortality rate in the Tissucol Duo S treated group compared to the control group. RESULTS: Mean age was 66.6+/-9.4 years, 76.3% of the patients were male. There was an increased 30-day-mortality rate in the Tissucol Duo S group compared to the control group (8.5 vs 3.5%, p<0.001). In order to determine if and to what extent the apparent fibrin effect might be due to confounding effects from covariates, an adjustment for potential confounding was done. However, multivariable adjustment did not reduce the risk of fibrin glue below an odds ratio of 2.2. CONCLUSION: Although the apparent increase in mortality risk associated with the use of fibrin glue could not be eliminated statistically, we consider Tissucol Duo S fibrin glue a safe and effective therapeutic tool in CABG surgery when it is applied correctly. Due to the retrospective character of this study some detailed information about the indication for the use of fibrin glue and its application is missing which may be important cofactors for mortality. For further clarification a prospective randomized study may be useful.

Succession of transiently active tumor-initiating cell clones in human pancreatic cancer xenografts.

Although tumor-initiating cell (TIC) self-renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long-term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self-renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.