Selection Of Subpopulations Research Articles

Differentiation-state plasticity is a targetable resistance mechanism in basal-like breast cancer

Intratumoral heterogeneity in cancers arises from genomic instability and epigenomic plasticity and is associated with resistance to cytotoxic and targeted therapies. We show here that cell-state heterogeneity, defined by differentiation-state marker expression, is high in triple-negative and basal-like breast cancer subtypes, and that drug tolerant persister (DTP) cell populations with altered marker expression emerge during treatment with a wide range of pathway-targeted therapeutic compounds. We show that MEK and PI3K/mTOR inhibitor-driven DTP states arise through distinct cell-state transitions rather than by Darwinian selection of preexisting subpopulations, and that these transitions involve dynamic remodeling of open chromatin architecture. Increased activity of many chromatin modifier enzymes, including BRD4, is observed in DTP cells. Co-treatment with the PI3K/mTOR inhibitor BEZ235 and the BET inhibitor JQ1 prevents changes to the open chromatin architecture, inhibits the acquisition of a DTP state, and results in robust cell death in vitro and xenograft regression in vivo.

PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil.

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs.

Design and estimation in clinical trials with subpopulation selection.

Population heterogeneity is frequently observed among patients' treatment responses in clinical trials because of various factors such as clinical background, environmental, and genetic factors. Different subpopulations defined by those baseline factors can lead to differences in the benefit or safety profile of a therapeutic intervention. Ignoring heterogeneity between subpopulations can substantially impact on medical practice. One approach to address heterogeneity necessitates designs and analysis of clinical trials with subpopulation selection. Several types of designs have been proposed for different circumstances. In this work, we discuss a class of designs that allow selection of a predefined subgroup. Using the selection based on the maximum test statistics as the worst‐case scenario, we then investigate the precision and accuracy of the maximum likelihood estimator at the end of the study via simulations. We find that the required sample size is chiefly determined by the subgroup prevalence and show in simulations that the maximum likelihood estimator for these designs can be substantially biased.

Why Do Herbivorous Mites Suppress Plant Defenses?

Plants have evolved numerous defensive traits that enable them to resist herbivores. In turn, this resistance has selected for herbivores that can cope with defenses by either avoiding, resisting or suppressing them. Several species of herbivorous mites, such as the spider mites Tetranychus urticae and Tetranychus evansi, were found to maximize their performance by suppressing inducible plant defenses. At first glimpse it seems obvious why such a trait will be favored by natural selection. However, defense suppression appeared to readily backfire since mites that do so also make their host plant more suitable for competitors and their offspring more attractive for natural enemies. This, together with the fact that spider mites are infamous for their ability to resist (plant) toxins directly, justifies the question as to why traits that allow mites to suppress defenses nonetheless seem to be relatively common? We argue that this trait may facilitate generalist herbivores, like T. urticae, to colonize new host species. While specific detoxification mechanisms may, on average, be suitable only on a narrow range of similar hosts, defense suppression may be more broadly effective, provided it operates by targeting conserved plant signaling components. If so, resistance and suppression may be under frequency-dependent selection and be maintained as a polymorphism in generalist mite populations. In that case, the defense suppression trait may be under rapid positive selection in subpopulations that have recently colonized a new host but may erode in relatively isolated populations in which host-specific detoxification mechanisms emerge. Although there is empirical evidence to support these scenarios, it contradicts the observation that several of the mite species found to suppress plant defenses actually are relatively specialized. We argue that in these cases buffering traits may enable such mites to mitigate the negative side effects of suppression in natural communities and thus shield this trait from natural selection.

Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip.

Protocols have been established to direct the differentiation of human induced pluripotent stem (iPS) cells into nephron progenitor cells and organoids containing many types of kidney cells, but it has been difficult to direct the differentiation of iPS cells to form specific types of mature human kidney cells with high yield. Here, we describe a detailed protocol for the directed differentiation of human iPS cells into mature, post-mitotic kidney glomerular podocytes with high (>90%) efficiency within 26 d and under chemically defined conditions, without genetic manipulations or subpopulation selection. We also describe how these iPS cell-derived podocytes may be induced to form within a microfluidic organ-on-a-chip (Organ Chip) culture device to build a human kidney Glomerulus Chip that mimics the structure and function of the kidney glomerular capillary wall in vitro within 35 d (starting with undifferentiated iPS cells). The podocyte differentiation protocol requires skills for culturing iPS cells, and the development of a Glomerulus Chip requires some experience with building and operating microfluidic cell culture systems. This method could be useful for applications in nephrotoxicity screening, therapeutic development, and regenerative medicine, as well as mechanistic study of kidney development and disease.

CD105 is regulated by hsa-miR-1287 and its expression is inversely correlated with osteopotential in SHED

A more accurate understanding of the molecular mechanisms and signaling pathways underpinning human mesenchymal stem cell (MSC) plasticity and differentiation properties is pivotal for accomplishing solid and diligent translation of MSC-based experimental therapeutics and clinical trials to broad clinical practice. In addition, this knowledge enables selection of MSC subpopulations with increased differentiation potential and/or use of exogenous factors to boost this potential. Here, we report that CD105 (ENG) is a predictive biomarker of osteogenic potential in two types of MSCs: stem cells from human exfoliated deciduous teeth (SHED) and human adipose-derived stem cells (hASC). We also validate that CD105 can be used to select and enrich for subpopulations of SHED and hASC with higher in vitro osteogenic potential. In addition, we show that hsa-mir-1287 regulates CD105 expression, and propose that fine-tuning hsa-mir-1287 levels could be used to control osteopotential in SHED. These findings provide better discernment of the molecular bases behind MSC osteogenic plasticity and open up new perspectives to leverage osteogenic potential in MSCs by modulation of a specific miRNA.

Safety and efficacy of nintedanib for the treatment of metastatic colorectal cancer

ABSTRACTIntroduction: Nintedanib (BIBF 1200) is an oral tyrosine kinase inhibitor that targets the vascular endothelial growth factor (VEGFR), platelet-derived growth factor (PDGFR) and fibroblast growth factor (FGFR) receptors. It is approved in Europe in combination with docetaxel for patients with advanced lung adenocarcinoma who have progressed to first-line chemotherapy. However, its role in the treatment of metastatic colorectal cancer (mCRC) is uncertain. Recent results from the LUME-Colon 1 pivotal phase III trial showed only a marginal increase in progression free survival over placebo in refractory mCRC patients, with a toxicity profile similar to other antiangiogenic agents, and no benefit in overall survival.Areas covered: The aim of this review is to summarize the pharmacology, efficacy and safety profile of nintedanib in the context of mCRC, and to provide some perspective regarding the role of this drug in clinical practice.Expert commentary: Nintedanib provides limited clinical benefit in refractory CRC and its use in this clinical setting is not warranted. Efforts shall continue to pursue the identification of predictive biomarkers that allow the selection of subpopulations with a greater likelihood to benefit from this therapeutic approach, in order to improve the benefit-risk and cost-benefit ratios of this and other antiangiogenic agents.

Abstract 4106: Mechanisms of secondary resistance to dual PI3K/mTOR inhibition in sarcomas with complex genetics

Abstract Background: Soft tissue sarcomas are a heterogeneous group of malignant tumors including more than 70 histological subtypes. We have recently demonstrated that dual targeting of PI3K/mTOR pathway is associated with significant anti-tumor activity in in vitro and in vivo models of leiomyosarcoma (LMS), one of the most frequent sarcoma subtype. Secondary resistance limits the efficacy of all targeted therapies. We have developed and characterized resistance models to a dual PI3K/mTOR inhibitor in order to describe underlying resistance mechanisms and develop alternative strategies. Methods: To develop in vitro BEZ-235 (dual PI3K/mTOR inhibitor) resistance models, 3 LMS and 1 myxofibrosarcoma cell lines were exposed to increasing doses of BEZ-235 several months. To characterize the resistance models, sensitivity to BEZ-235 was assessed by MTT assay and Annexin V-PI staining. To test in vivo the resistance models, effects of BEZ-235 treatment were observed in mice with subcutaneous cell line-derived xenograft tumors for 3 weeks. The transcriptome profile of sensitive and resistant tumors to BEZ-235 was analyzed by using RNA-seq. Results: After prolonged exposure of cell lines to BEZ-235, we obtained secondary resistant cell lines characterized by an IC50 value 5- to 21-fold higher than parental sensitive cells. Resistant cells were also significantly less sensitive to BEZ-235-induced apoptosis than parental cells. We confirmed the resistance patterns in vivo with a significantly higher anti-tumor effect of BEZ235 in sensitive cell-line derived xenograft group compared with resistant cell-line derived xenograft group. Deep transcriptome profiling indicated overexpression of stem cells related genes in resistant cells as well as a modification of the cellular metabolism. By using 1) RNAi-mediated suppression of pyruvate carboxylase (PC), a potential key player in our resistance models and 2) hallmarks of CSC like CD133 and enhanced aldehyde dehydrogenase (ALDH) activity, we confirmed that resistant tumors were characterized by a significantly higher proportion of cancer stem cells and by a metabolic shift with a strong deregulation of glucose metabolism. Conclusions: Selection of CSC subpopulation and metabolism shift play a crucial role in secondary resistance to dual PI3K/mTOR inhibition in sarcoma. Strategies assessing anti-CSC agents to suppress acquired resistance may have major implications to improve efficacy of dual PI3K/mTOR targeting in human malignancies. Citation Format: Benjamin Fourneaux, Aurélien Bourdon, Vanessa Chaire, Carlo Lucchesi, Marie Karanian, Raphael Pineau, Audrey Laroche, Antoine Italiano. Mechanisms of secondary resistance to dual PI3K/mTOR inhibition in sarcomas with complex genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4106. doi:10.1158/1538-7445.AM2017-4106

Management of lung cancer brain metastasis: An overview

AbstractWith the improvements in systemic treatment for lung cancer, distant metastasis to sanctuary sites such as brain has become an increasingly more important issue. The management of these patients consists of supportive care and disease-directed treatment. Combined modality treatment (surgical resection or radiosurgery, followed by whole brain radiotherapy) of brain metastases has greatly improved the local control of disease in patients with single lesion, good functional performance status, and controlled extracranial disease as demonstrated in prospective randomized studies. For patients with multiple brain metastases, conventional fractionated whole brain radiotherapy continues to be a standard and efficacious treatment. At present, experience with the use of molecularly targeted tyrosine kinase inhibitors in nonsmall cell lung cancer patients with activating mutations in the epidermal growth factor receptor gene and anaplastic lymphoma kinase gene is growing. However, their effectiveness in patients with brain metastases is not well established. In the arena of targeted therapies, vascular endothelial growth factor pathway inhibitors such as bevacizumab have shown some activity in brain metastases. Further prospective studies are necessary to facilitate selection of patient subpopulation for targeted agents in future studies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Cloning of CHO Cells, Productivity and Genetic Stability—A Discussion

While many perceive mammalian cell culture-based manufacturing for biopharmaceuticals an established technology, numerous open questions remain to be solved. Genetic diversity and mutation rates in CHO cells have been underestimated since progeny of a clonal CHO cell become genetically diverse with each cell division. This is an important issue since products are made in bioreactors containing up to 1014 cells that have divided for weeks. Regulatory interest in “Proof of Clonality” is a misguided and misunderstood concern in this context. We revisit decades of research on scope and rate of genetic changes in CHO cells and suggest approaches to minimize trends for genomic instability when establishing reliable manufacturing processes. A concept is proposed for transfection-derived cell populations containing relatively stable (The term “stable” will always be used in a context of relative stability—considering time frames of weeks to months at best.) “CHO master sequence” genomes (containing the desired DNAs of interest). Stable cell populations are to be selected for and maintained for the various phases of manufacturing under specific culture conditions reducing trends for the selection of diverse subpopulations. Such conditions are based on insights gained from population genetics, evolutionary landscape fitness principles, and a 40-year old model for evolution of error prone replicating systems—the Quasi-Species concept.

NATURAL ANTIBIOTIC RESISTANCE GENES IN SOIL BACTERIA AND INFLUENCE OF ORGANIC FERTILISERS ON THEIR PREVALENCE AND HORIZONTAL TRANSFER

All natural antibiotics available to modern medicine are products of soil-dwelling bacteria and fungi. In addition, all resistance genes which are being detected in human pathogens existed in soil bacteria even before antibiotics were discovered and brought into use. However, the concentrations of natural antibiotics in soil are usually subinhibitory – insuffi cient for the selection of resistant subpopulations of microorganisms. The consumption of organic fertilisers for agricultural soil amendment increases proportionally to the consumers’ growing demand for organically produced food. Manure originating from industrial pig, cattle and poultry farms is not only the source of nutrients which stimulate the vital functions of soil microorganisms, but also of antibiotics and bacteria harbouring various resistance mechanisms. The application of organic fertilizer leads to disruption of the natural balance between bacterial communities in the soil through several mechanisms, and infl uences the increase in the prevalence of resistance genes and promotes their horizontal transfer. Whether as-yet-unknown resistance genes in soil bacteria may pose threat to human health if transferred from commensal bacteria in the environment to pathogen species, or migrate to clinical settings via food chain or in some other possible route - remains an open question.

Susceptibility and PK/PD relationships of Staphylococcus aureus strains isolated from the milk of sheep and goats with clinical mastitis to five veterinary fluoroquinolones

Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of veterinary fluoroquinolones as enrofloxacin, its metabolite ciprofloxacin, danofloxacin, difloxacin and marbofloxacin against Staphylococcus aureus strains (n=24) isolated from milk of...

Rapid adaptation drives invasion of airway donor microbiota by Pseudomonas after lung transplantation

In cystic fibrosis (CF) patients, chronic airway infection by Pseudomonas leads to progressive lung destruction ultimately requiring lung transplantation (LT). Following LT, CF-adapted Pseudomonas strains, potentially originating from the sinuses, may seed the allograft leading to infections and reduced allograft survival. We investigated whether CF-adapted Pseudomonas populations invade the donor microbiota and adapt to the non-CF allograft. We collected sequential Pseudomonas isolates and airway samples from a CF-lung transplant recipient during two years, and followed the dynamics of the microbiota and Pseudomonas populations. We show that Pseudomonas invaded the host microbiota within three days post-LT, in association with a reduction in richness and diversity. A dominant mucoid and hypermutator mutL lineage was replaced after 11 days by non-mucoid strains. Despite antibiotic therapy, Pseudomonas dominated the allograft microbiota until day 95. We observed positive selection of pre-LT variants and the appearance of novel mutations. Phenotypic adaptation resulted in increased biofilm formation and swimming motility capacities. Pseudomonas was replaced after 95 days by a microbiota dominated by Actinobacillus. In conclusion, mucoid Pseudomonas adapted to the CF-lung remained able to invade the allograft. Selection of both pre-existing non-mucoid subpopulations and of novel phenotypic traits suggests rapid adaptation of Pseudomonas to the non-CF allograft.

Adaptation of CHO cells in serum-free conditions for erythropoietin production: Application of EVOP technique for process optimization.

Mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. Usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. However, due to scientific and regulatory concerns, serum-free conditions are required in recombinant protein production. Cell lines that are intended for commercial recombinant protein production have to adapt to serum- or protein-free conditions early in their development. This is a labor- and time-consuming process because of the specific cell requirements related to their adaptation in new microenvironment. In the present study, a Chinese hamster ovary (CHO) cell line producing glycosylated recombinant human erythropoietin (rhEPO) was adapted for growth and rhEPO production in serum- and protein-free conditions. The physiology, growth parameters, and morphology of the CHO cells and rhEPO biosynthesis and structure were closely monitored during the adaptation process to avoid unwanted selection of cell subpopulations. The results showed that the CHO cells were successfully adapted to suspension growth and rhEPO production in the protein-free conditions and that the structure of rhEPO remained nearly unchanged. In addition, during rhEPO production in the protein-free suspension conditions, the agitation rate seem to be significant for optimal process performance in contrast to the initial cell concentration, evaluated through evolutionary operation method.

Comparison of different clinical development plans for confirmatory subpopulation selection

Given ever increasing costs to develop a new drug and intense competition, adaptive enrichment designs are an attractive option for a development program that allows selecting a potential subgroup defined by a binary biomarker. Such designs explicitly factor in the possibility that the new drug might differentially benefit distinct biomarker subgroups. We have compared three clinical development plans for a time-to-event endpoint, such as overall survival, that all lead to a decision in a pivotal trial either in all comers only, in allcomers and biomarker positive, in the biomarker positive only, or to declare the drug futile. The decision about which hypothesis to test at the final analysis is made based on a fast time-to-event endpoint, such as progression-free survival, at an interim analysis. We quantify the time gain when using an adaptive enrichment Phase II/III design versus alternative development approaches and we outline what type of biomarker needs to be available prior to Phase II in each scenario. We conclude with a discussion of further features of each of the considered development plans.

Host-Parasite Interactions in Chagas Disease: Genetically Unidentical Isolates of a Single Trypanosoma cruzi Strain Identified In Vitro via LSSP-PCR.

The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of Trypanosoma cruzi is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of Trypanosoma cruzi Berenice-78 and characterized via LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and in vitro profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct Trypanosoma cruzi isolates may be obtained upon experimental murine infection with a single polyclonal Trypanosoma cruzi strain.

Abstract A31: Combination therapies guided by evolution of tumor heterogeneity.

Abstract Successful treatment of hematologic malignancies is challenged by genetic heterogeneity of tumors and the dynamic evolution of the heterogeneity in response to targeted drugs as well as chemotherapeutics. We are applying an integrated computational/experimental approach to elucidating principles which can guide selection of effective combination therapies by mathematical analysis of how tumors evolve with respect to drug sensitivity and resistance during treatment. In an initial pharmacological screen of targeted drugs and chemotherapeutics on murine Bcr-Abl; p19Arf-/- ALL cells, we found that crizotinib was particularly effective (IC50 = ~500 nM) with comparable efficacy to that for imatinib (IC50 = ~200 nM). Moreover, in a derivative cell line bearing spontaneous Bcr-AblT315I mutation, crizotinib exhibited efficacy comparable to that of the Bcr-AblWT parental cell line. This same Bcr-AblT315I cell line showed strong resistance to Abl inhibitors imatinib, dasatinib, nilotinib, and bosutinib. Imatinib and crizotinib also exhibited comparable in vivo efficacy, based on overall survival of syngeneic immunocompetent recipient mice transplanted with Bcr-Abl; p19Arf-/- ALL cells. Cell cycle profiles and signaling analyses suggest that crizotinib induces G2/M arrest and subsequently bim-dependent caspase-mediated apoptosis. This phenotype has been subsequently validated in the human K562 Ph+ CML and murine Baf3 Bcr-AblWT and Bcr-AblT315I cell lines. Resistant populations were also derived through dose escalation treatments with imatinib, dasatinib, nilotinib, foretinib, and crizotinib. Bcr-Abl; p19Arf-/- ALL cells were treated at IC90, and recovered populations were subsequently split into: [a] no-drug media, with sensitivity to different sets of drugs assessed at specific time points post-recovery; and [b] new media with drug concentration at 2x the previous dose (e.g., IC90 2x). Cell populations that were resistant to dasatinib at 1x and 2x IC90 became even more sensitive to crizotinib and foretinib. The drug sensitivity does not appear to be a transient behavior as a result of intracellular rewiring, but rather a change in the tumor heterogeneity via selection of particular subpopulations, as evidenced by sustained sensitivity when these populations were analyzed 24h, 48h, 72h, 5 days, and 9 days post drug selection in no-drug media. The sensitivity was abrogated at IC90 4x and above. Cell populations resistant at these higher doses of dasatinib also became cross-resistant to imatinib and foretinib. This potentially suggests particular stages in the tumor evolution under continued drug selection for which they are extremely sensitive to other pharmacological agents. These findings suggest a novel combination treatment strategy taking into account the evolutionary trajectories of tumor heterogeneity, with drugs dosed sequentially in a multi-course regimen. The sequential treatment is not to exploit vulnerabilities of intracellular rewiring induced by the first drug, but rather vulnerabilities of resulting expanded subpopulations upon selection by the first drug. Citation Format: Boyang Zhao, Michael T. Hemann, Douglas A. Lauffenburger. Combination therapies guided by evolution of tumor heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A31.

Mutational status of naevus-associated melanomas.

The origin of melanoma has always been a debated subject, as well as the role of adjacent melanocytic naevi. Epidemiological and histopathological studies point to melanomas arising either de novo or from a naevus. To evaluate the presence of mutations in genes from well-known melanomagenesis pathways in a large series of naevus-associated melanomas. Sixty-one melanomas found in association with a pre-existing naevus were microdissected, after careful selection of cell subpopulations, and submitted to Sanger sequencing of the BRAF, NRAS, c-KIT, PPP6C, STK19 and RAC1 genes. Each gene was evaluated twice in all samples by sequencing or by sequencing and another confirmation method, allele-specific fluorescent polymerase chain reaction (PCR) and capillary electrophoresis detection or by SNaPshot analysis. Only mutations confirmed via two different molecular methods or twice by sequencing were considered positive. The majority of cases presented concordance of mutational status between melanoma and the associated naevus for all six genes (40 of 60; 66.7%). Nine cases presented concomitant BRAF and NRAS mutations, including one case in which both the melanoma and the adjacent naevus harboured V600E and Q61K double mutations. In two cases, both melanoma and associated naevus located on acral sites were BRAF mutated, including an acral lentiginous melanoma. To our knowledge this is the largest naevus-associated melanoma series evaluated molecularly. The majority of melanomas and adjacent naevi in our sample share the same mutational profile, corroborating the theory that the adjacent naevus and melanoma are clonally related and that the melanoma originated within a naevus.

Estimation after subpopulation selection in adaptive seamless trials.

During the development of new therapies, it is not uncommon to test whether a new treatment works better than the existing treatment for all patients who suffer from a condition (full population) or for a subset of the full population (subpopulation). One approach that may be used for this objective is to have two separate trials, where in the first trial, data are collected to determine if the new treatment benefits the full population or the subpopulation. The second trial is a confirmatory trial to test the new treatment in the population selected in the first trial. In this paper, we consider the more efficient two‐stage adaptive seamless designs (ASDs), where in stage 1, data are collected to select the population to test in stage 2. In stage 2, additional data are collected to perform confirmatory analysis for the selected population. Unlike the approach that uses two separate trials, for ASDs, stage 1 data are also used in the confirmatory analysis. Although ASDs are efficient, using stage 1 data both for selection and confirmatory analysis introduces selection bias and consequently statistical challenges in making inference. We will focus on point estimation for such trials. In this paper, we describe the extent of bias for estimators that ignore multiple hypotheses and selecting the population that is most likely to give positive trial results based on observed stage 1 data. We then derive conditionally unbiased estimators and examine their mean squared errors for different scenarios.©2015 The Authors. Statistics in Medicine Published by JohnWiley & Sons Ltd.