Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH.

Comparative genomic hybridization by means of BAC microarrays (array CGH) allows high-resolution profiling of copy-number aberrations in tumor DNA. However, specific genetic lesions associated with small but clinically relevant tumor areas may pass undetected due to intra-tumor heterogeneity and/or the presence of contaminating normal cells. Here, we show that the combination of laser capture microdissection, phi29 DNA polymerase-mediated isothermal genomic DNA amplification, and array CGH allows genomic profiling of very limited numbers of cells. Moreover, by means of simple statistical models, we were able to bypass the exclusion of amplification distortions and variability prone areas, and to detect tumor-specific chromosomal gains and losses. We applied this new combined experimental and analytical approach to the genomic profiling of colorectal adenomatous polyps and demonstrated our ability to accurately detect single copy gains and losses affecting either whole chromosomes or small genomic regions from as little as 2 ng of DNA or 1000 microdissected cells

Combination Early-Phase Trials of Anticancer Agents in Children and Adolescents

PURPOSEThere is an increasing need to evaluate innovative drugs for childhood cancer using combination strategies. Strong biological rationale and clinical experience suggest that multiple agents will be more efficacious than monotherapy for most diseases and may overcome resistance mechanisms and increase synergy. The process to evaluate these combination trials needs to maximize efficiency and should be agreed by all stakeholders.METHODSAfter a review of existing combination trial methodologies, regulatory requirements, and current results, a consensus among stakeholders was achieved.RESULTSCombinations of anticancer therapies should be developed on the basis of mechanism of action and robust preclinical evaluation, and may include data from adult clinical trials. The general principle for combination early-phase studies is that, when possible, clinical trials should be dose- and schedule-confirmatory rather than dose-exploratory, and every effort should be made to optimize doses early. Efficient early-phase combination trials should be seamless, including dose confirmation and randomized expansion. Dose evaluation designs for combinations depend on the extent of previous knowledge. If not previously evaluated, limited evaluation of monotherapy should be included in the same clinical trial as the combination. Randomized evaluation of a new agent plus standard therapy versus standard therapy is the most effective approach to isolate the effect and toxicity of the novel agent. Platform trials may be valuable in the evaluation of combination studies. Patient advocates and regulators should be engaged with investigators early in a proposed clinical development pathway and trial design must consider regulatory requirements.CONCLUSIONAn optimized, agreed approach to the design and evaluation of early-phase pediatric combination trials will accelerate drug development and benefit all stakeholders, most importantly children and adolescents with cancer.</p

A Targeted Constitutive Mutation in the Apc Tumor Suppressor Gene Underlies Mammary But Not Intestinal Tumorigenesis

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc+/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc+/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion

Contribution of SARS-CoV-2 infection preceding COVID-19 mRNA vaccination to generation of cellular and humoral immune responses in children

Primary COVID-19 vaccination for children, 5-17 years of age, was offered in the Netherlands at a time when a substantial part of this population had already experienced a SARS-CoV-2 infection. While vaccination has been shown effective, underlying immune responses have not been extensively studied. We studied immune responsiveness to one and/or two doses of primary BNT162b2 mRNA vaccination and compared the humoral and cellular immune response in children with and without a preceding infection. Antibodies targeting the original SARS-CoV-2 Spike or Omicron Spike were measured by multiplex immunoassay. B-cell and T-cell responses were investigated using enzyme-linked immunosorbent spot (ELISpot) assays. The activation of CD4+ and CD8+ T cells was studied by flowcytometry. Primary vaccination induced both a humoral and cellular adaptive response in naive children. These responses were stronger in those with a history of infection prior to vaccination. A second vaccine dose did not further boost antibody levels in those who previously experienced an infection. Infection-induced responsiveness prior to vaccination was mainly detected in CD8+ T cells, while vaccine-induced T-cell responses were mostly by CD4+ T cells. Thus, SARS-CoV-2 infection prior to vaccination enhances adaptive cellular and humoral immune responses to primary COVID-19 vaccination in children. As most children are now expected to contract infection before the age of five, the impact of infection-induced immunity in children is of high relevance. Therefore, considering natural infection as a priming immunogen that enhances subsequent vaccine-responsiveness may help decision-making on the number and timing of vaccine doses

BAC CGH array profiling using Phi29-amplified microdissected samples

Details of the experiment are available within the Nucleic Acid Research, 2004 manuscript "Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH", by Cardoso J. et al

BAC CGH array profiling using Phi29-amplified microdissected samples

Details of the experiment are available within the Nucleic Acid Research, 2004 manuscript "Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH", by Cardoso J. et al

Overview of the phenotypic comparisons between <i>Apc</i>^+/1572T and other <i>Apc</i>-mutant mouse models.

<p>Notes: Incidence is given as percentage of affected animals.</p>1,3<p>Animals were sacrificed when signs of discomfort were apparent and/or when tumor size reached 2 cm.</p>2<p>The incidence of GI tumors was calculated after exclusion of the pyloric lesions as these present in clusters often difficult to count.</p>3<p>The multiplicity of GI tumors was calculated based on all animals with the exception of those where the high tumor burden made the count not feasible.</p>*<p>This specific animal was found to carry a single tumor at 21 months of age, likely to represent a sporadic case. Background (B) of the different strains analyzed: F1: C57Bl6/J x 129Ola; Ola: inbred 129Ola; B6: inbred C57Bl6/J. n.d. not determined.</p

Biochemical characterization of the targeted <i>Apc</i>1572T allele.

<p>(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the <i>Apc</i>1572T, <i>Apc</i>1638N, and <i>Apc</i>1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of <i>Apc</i>^1638N/1638N ES lines <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Kielman1" target="_blank">[5]</a>. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of <i>Apc</i>^+/+ (1) and <i>Apc</i>-mutant ES cell lines: Apc^1638T/1638T (2); Apc^1572T/1572T (3); Apc^1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Korinek1" target="_blank">[10]</a> together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in <i>Ap</i>c-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.</p

Phenotypic and molecular analysis of the compound <i>in cis Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mouse model.

<p>(A) Schematic illustration of the chr. 18 LOH event in intestinal tumors from <i>in cis Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice leading to loss of both <i>Smad4</i> and <i>Apc</i> wild-type alleles. (B) Comparative phenotypic analysis of the intestinal and mammary tumor predisposition among <i>Apc</i>^+/1572T, <i>Smad4</i>^+/Sad, and <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice. <i>Notes</i>: (1) The incidence of GI tumors was calculated after exclusion of the pyloric lesions as these present in clusters often difficult to count. (2) The multiplicity of GI tumors was calculated based on all animals with the exception of those where the high tumor burden made the count not feasible. The asterisks indicate that the apparent absence of intestinal tumor in <i>Smad4</i>^+/Sad control animals is not in contradiction with what previously published. These mice were sacrificed at time points matched with the ages at which compound <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice had to be sacrificed due to the high GI and mammary tumor burden (♀: 90.4 days +/−28.4; ♂: 118.5 days +/−26.2). However, in <i>Smad4</i>^+/Sad animals the majority of the tumors appear at 9 months of age <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Alberici1" target="_blank">[15]</a>. (C) H&E staining of intestinal tumor sections from <i>Apc</i>^+/1572T (top), <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad (middle), and <i>Smad4</i>^+/Sad (bottom) mice. (D) Smad4 IHC analysis of two intestinal adenomas from <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice showing loss of Smad4 expression. LOH was observed in 100% of the polyps (n = 15) analyzed. PCR–based LOH analysis of the same cohort of <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad polyps revealed loss of wild-type <i>Apc</i> allele in 87% of the cases (13/15; data not shown).</p