The European MAPPYACTS Trial: Precision Medicine Program in Pediatric and Adolescent Patients with Recurrent Malignancies.

ABSTRACT: MAPPYACTS (NCT02613962) is an international prospective precision medicine trial aiming to define tumor molecular profiles in pediatric patients with recurrent/refractory malignancies in order to suggest the most adapted salvage treatment. From February 2016 to July 2020, 787 patients were included in France, Italy, Ireland, and Spain. At least one genetic alteration leading to a targeted treatment suggestion was identified in 436 patients (69%) with successful sequencing; 10% of these alterations were considered "ready for routine use." Of 356 patients with follow-up beyond 12 months, 107 (30%) received one or more matched targeted therapies-56% of them within early clinical trials-mainly in the AcSé-ESMART platform trial (NCT02813135). Overall, matched treatment resulted in a 17% objective response rate, and of those patients with ready for routine use alterations, it was 38%. In patients with extracerebral tumors, 76% of actionable alterations detected in tumor tissue were also identified in circulating cell-free DNA (cfDNA). SIGNIFICANCE: MAPPYACTS underlines the feasibility of molecular profiling at cancer recurrence in children on a multicenter, international level and demonstrates benefit for patients with selected key drivers. The use of cfDNA deserves validation in prospective studies. Our study highlights the need for innovative therapeutic proof-of-concept trials that address the underlying cancer complexity. This article is highlighted in the In This Issue feature, p. 1171.

Molecular diagnosis of retinoblastoma by circulating tumor DNA analysis.

PURPOSE: The analysis of circulating tumor DNA (ctDNA), a fraction of total cell-free DNA (cfDNA), might be of special interest in retinoblastoma patients. Because the accessibility to tumor tissue is very limited in these patients, either for histopathological diagnosis of suspicious intraocular masses (biopsies are proscribed) or for somatic RB1 studies and genetic counseling (due to current successful conservative approaches), we aim to validate the detection of ctDNA in plasma of non-hereditary retinoblastoma patients by molecular analysis of RB1 gene. EXPERIMENTAL DESIGN: In a cohort of 19 intraocular unilateral non-hereditary retinoblastoma patients for whom a plasma sample was available at diagnosis, we performed high-deep next-generation sequencing (NGS) of RB1 in cfDNA. Two different bioinformatics/statistics approaches were applied depending on whether the somatic RB1 status was available or not. RESULTS: Median plasma sample volume was 600 µL [100-1000]; median cfDNA plasma concentration was 119 [38-1980] and 27 [11-653] ng/mL at diagnosis and after complete remission, respectively. In the subgroup of patients with known somatic RB1 alterations (n = 11), seven of nine somatic mutations were detected (median allele fraction: 6.7%). In patients without identified somatic RB1 alterations (n = 8), six candidate variants were identified for seven patients. CONCLUSIONS: Despite small tumor size, blood-ocular barrier, poor ctDNA blood release and limited plasma sample volumes, we confirm that it is possible to detect ctDNA with high-deep NGS in plasma from patients with intraocular non-hereditary retinoblastoma. This may aid in diagnosis of suspicious cases, family genetic counseling or follow-up of residual intraocular disease.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1).

PURPOSE: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

Molecular features of untreated breast cancer and initial metastatic event inform clinical decision-making and predict outcome: long-term results of ESOPE, a single-arm prospective multicenter study.

BACKGROUND: Prognosis evaluation of advanced breast cancer and therapeutic strategy are mostly based on clinical features of advanced disease and molecular profiling of the primary tumor. Very few studies have evaluated the impact of metastatic subtyping during the initial metastatic event in a prospective study. The genomic landscape of metastatic breast cancer has mostly been described in very advanced, pretreated disease, limiting the findings transferability to clinical use. METHODS: We developed a multicenter, single-arm, prospective clinical trial in order to address these issues. Between November 2010 and September 2013, 123 eligible patients were included. Patients at the first, untreated metastatic event were eligible. All matched primary tumors and metastatic samples were centrally reviewed for pathological typing. Targeted and whole-exome sequencing was applied to matched pairs of frozen tissue. A multivariate overall survival analysis was performed (median follow-up 64 months). RESULTS: Per central review in 84 patients (out of 130), we show that luminal A breast tumors are more prone to subtype switching. By combining targeted sequencing of a 91 gene panel (n = 67) and whole-exome sequencing (n = 30), a slight excess of mutations is observed in the metastases. Luminal A breast cancer has the most heterogeneous mutational profile and the highest number of mutational signatures, when comparing primary tumor and the matched metastatic tissue. Tumors with a subtype change have more mutations that are private. The metastasis-specific mutation load is significantly higher in late than in de novo metastases. The most frequently mutated genes were TP53 and PIK3CA. The most frequent metastasis-specific druggable genes were PIK3CA, PTEN, KDR, ALK, CDKN2A, NOTCH4, POLE, SETD2, SF3B1, and TSC2. Long-term outcome is driven by a combination of tumor load and metastasis biology. CONCLUSIONS: Profiling of the first, untreated, metastatic event of breast cancer reveals a profound heterogeneity mostly in luminal A tumors and in late metastases. Based on this profiling, we can derive information relevant to prognosis and therapeutic intervention, which support current guidelines recommending a biopsy at the first metastatic relapse. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov ( NCT01956552 ).

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

Study of chromatin remodeling genes implicates SMARCA4 as a putative player in oncogenesis in neuroblastoma.

In neuroblastoma (NB), genetic alterations in chromatin remodeling (CRGs) and epigenetic modifier genes (EMGs) have been described. We sought to determine their frequency and clinical impact. Whole exome (WES)/whole genome sequencing (WGS) data and targeted sequencing (TSCA®) of exonic regions of 33 CRGs/EMGs were analyzed in tumor samples from 283 NB patients, with constitutional material available for 55 patients. The frequency of CRG/EMG variations in NB cases was then compared to the Genome Aggregation Database (gnomAD). The sequencing revealed SNVs/small InDels or focal CNAs of CRGs/EMGs in 20% (56/283) of all cases, occurring at a somatic level in 4 (7.2%), at a germline level in 12 (22%) cases, whereas for the remaining cases, only tumor material could be analyzed. The most frequently altered genes were ATRX (5%), SMARCA4 (2.5%), MLL3 (2.5%) and ARID1B (2.5%). Double events (SNVs/small InDels/CNAs associated with LOH) were observed in SMARCA4 (n = 3), ATRX (n = 1) and PBRM1 (n = 1). Among the 60 variations, 24 (8.4%) targeted domains of functional importance for chromatin remodeling or highly conserved domains but of unknown function. Variations in SMARCA4 and ATRX occurred more frequently in the NB as compared to the gnomAD control cohort (OR = 4.49, 95%CI: 1.63-9.97, p = 0.038; OR 3.44, 95%CI: 1.46-6.91, p = 0.043, respectively). Cases with CRG/EMG variations showed a poorer overall survival compared to cases without variations. Genetic variations of CRGs/EMGs with likely functional impact were observed in 8.4% (24/283) of NB. Our case-control approach suggests a role of SMARCA4 as a player of NB oncogenesis.

Limited Sensitivity of Circulating Tumor DNA Detection by Droplet Digital PCR in Non-Metastatic Operable Gastric Cancer Patients.

This study was designed to monitor circulating tumor DNA (ctDNA) levels during perioperative chemotherapy in patients with non-metastatic gastric adenocarcinoma. Plasma samples were prospectively collected in patients undergoing perioperative chemotherapy for non-metastatic gastric adenocarcinoma (excluding T1N0) prior to the initiation of perioperative chemotherapy, before and after surgery (NCT02220556). In each patient, mutations retrieved by targeted next-generation sequencing (NGS) on tumor samples were then tracked in circulating cell-free DNA from 4 mL of plasma by droplet digital PCR. Thirty-two patients with a diagnosis of non-metastatic gastric adenocarcinoma were included. A trackable mutation was identified in the tumor in 20 patients, seven of whom experienced relapse during follow-up. ctDNA was detectable in four patients (N = 4/19, sensitivity: 21%; 95% confidence interval CI = 8.5⁻43%, no baseline plasma sample was available for one patient), with a median allelic frequency (MAF) of 1.6% (range: 0.8⁻2.3%). No patient with available plasma samples (N = 0/18) had detectable ctDNA levels before surgery. After surgery, one of the 13 patients with available plasma samples had a detectable ctDNA level with a low allelic frequency (0.7%); this patient experienced a very short-term distant relapse only 3 months after surgery. No ctDNA was detected after surgery in the other four patients with available plasma samples who experienced a later relapse (median = 14.4, range: 9.3⁻26 months). ctDNA monitoring during preoperative chemotherapy and after surgery does not appear to be a useful tool in clinical practice for non-metastatic gastric cancer to predict the efficacy of chemotherapy and subsequent relapse, essentially due to the poor sensitivity of ctDNA detection.

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

ART-DeCo: easy tool for detection and characterization of cross-contamination of DNA samples in diagnostic next-generation sequencing analysis.

Next-generation sequencing (NGS) is routinely used for constitutional genetic analysis. However, cross-contamination between samples constitutes a major risk that could impact the results of the analysis. We have developed ART-DeCo, a tool using the allelic ratio (AR) of the Single Nucleotide Polymorphisms sequenced with regions of interest. When a sample is contaminated by DNA with a different genotype, unexpected ARs are obtained, which are in turn used for detection of contamination with a screening test, followed by identification and quantification of the contaminant. Following optimization, ART-DeCo was applied to 2222 constitutional DNA samples. The screening test was positive for 191 samples. In 33 cases (contamination percentages: 1.3% to 29.2%), the contaminant was identified and was mostly located in adjacent wells. Three other positive cases were due to barcoding errors or mixture of two DNA samples. Interestingly, the last contaminated sample corresponded to a bone marrow transplant recipient. Lastly, no contaminant was identified in 154 weakly positive ( < 4%) samples that were considered to be irrelevant to constitutional genetic analysis. ART-DeCo lends itself to mandatory quality control procedures, also highlighting the delicate steps of library preparation, resulting in practice improvement. Importantly, ART-DeCo can be implemented in any NGS workflow, from gene panel to genome-wide analyses. https://sourceforge.net/projects/ngs-art-deco/ .

Targeted next-generation sequencing identifies clinically relevant somatic mutations in a large cohort of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of primary breast cancer. Using a custom-made breast cancer gene sequencing panel, we investigated somatic mutations in IBC to better understand the genomic differences compared with non-IBC and to consider new targeted therapy in IBC patients. METHODS: Targeted next-generation sequencing (NGS) of 91 candidate breast cancer-associated genes was performed on 156 fresh-frozen breast tumor tissues from IBC patients. Mutational profiles from 197 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as non-IBC controls for comparison analysis. The mutational landscape of IBC was correlated with clinicopathological data and outcomes. RESULTS: After genotype calling and algorithmic annotations, we identified 392 deleterious variants in IBC and 320 variants in non-IBC cohorts, respectively. IBC tumors harbored more mutations than non-IBC (2.5 per sample vs. 1.6 per sample, p < 0.0001). Eighteen mutated genes were significantly different between the two cohorts, namely TP53, CDH1, NOTCH2, MYH9, BRCA2, ERBB4, POLE, FGFR3, ROS1, NOTCH4, LAMA2, EGFR, BRCA1, TP53BP1, ESR1, THBS1, CASP8, and NOTCH1. In IBC, the most frequently mutated genes were TP53 (43.0%), PIK3CA (29.5%), MYH9 (8.3%), NOTCH2 (8.3%), BRCA2 (7.7%), ERBB4 (7.1%), FGFR3 (6.4%), POLE (6.4%), LAMA2 (5.8%), ARID1A (5.1%), NOTCH4 (5.1%), and ROS1 (5.1%). After grouping 91 genes on 10 signaling pathways, we found that the DNA repair pathway for the triple-negative breast cancer (TNBC) subgroup, the RTK/RAS/MAPK and cell cycle pathways for the HR-/HER2+ subgroup, the DNA repair, RTK/RAS/MAPK, and NOTCH pathways for the HR+/HER2- subgroup, and the DNA repair, epigenome, and diverse pathways for the HR+/HER2+ subgroup were all significantly differently altered between IBC and non-IBC. PIK3CA mutation was independently associated with worse metastasis-free survival (MFS) in IBC since the median MFS for the PIK3CA mutant type was 26.0 months and for the PIK3CA wild type was 101.1 months (p = 0.002). This association was observed in TNBC (p = 0.04) and the HR-/HER2+ subgroups (p = 0.0003), but not in the HR+/HER2- subgroup of IBC. CONCLUSIONS: Breast cancer-specific targeted NGS uncovered a high frequency of deleterious somatic mutations in IBC, some of which may be relevant for clinical management.

Relevance of a molecular tumour board (MTB) for patients' enrolment in clinical trials: experience of the Institut Curie.

BACKGROUND: High throughput molecular screening techniques allow the identification of multiple molecular alterations, some of which are actionable and can be targeted by molecularly targeted agents (MTA). We aimed at evaluating the relevance of using this approach in the frame of Institut Curie Molecular Tumor Board (MTB) to guide patients with cancer to clinical trials with MTAs. PATIENTS AND METHODS: We included all patients presented at Institut Curie MTB from 4 October 2014 to 31 October 2017. The following information was extracted from the chart: decision to perform tumour profiling, types of molecular analyses, samples used, molecular alterations identified and those which are actionable, and inclusion in a clinical trial with matched MTA. RESULTS: 736 patients were presented at the MTB. Molecular analyses were performed in 442 patients (60%). Techniques used included next-generation sequencing, comparative genomic hybridisation array and/or other techniques including immunohistochemistry in 78%, 51% and 58% of patients, respectively. Analyses were performed on a fresh frozen biopsy in 91 patients (21%), on archival tissue (fixed or frozen) in 326 patients (74%) and on both archival and fresh frozen biopsy in 25 patients (6%). At least one molecular alteration was identified in 280 analysed patients (63%). An actionable molecular alteration was identified in 207 analysed patients (47%). Forty-five analysed patients (10%) were enrolled in a clinical trial with matched MTA and 29 additional patients were oriented and included in a clinical trial based on a molecular alteration identified prior to the MTB analysis. Median time between date of specimen reception and molecular results was 28 days (range: 5-168). CONCLUSIONS: The implementation of an MTB at Institut Curie enabled the inclusion of 10% of patients into a clinical trial with matched therapy.

QuantumClone: clonal assessment of functional mutations in cancer based on a genotype-aware method for clonal reconstruction.

Motivation: In cancer, clonal evolution is assessed based on information coming from single nucleotide variants and copy number alterations. Nonetheless, existing methods often fail to accurately combine information from both sources to truthfully reconstruct clonal populations in a given tumor sample or in a set of tumor samples coming from the same patient. Moreover, previously published methods detect clones from a single set of variants. As a result, compromises have to be done between stringent variant filtering [reducing dispersion in variant allele frequency estimates (VAFs)] and using all biologically relevant variants. Results: We present a framework for defining cancer clones using most reliable variants of high depth of coverage and assigning functional mutations to the detected clones. The key element of our framework is QuantumClone, a method for variant clustering into clones based on VAFs, genotypes of corresponding regions and information about tumor purity. We validated QuantumClone and our framework on simulated data. We then applied our framework to whole genome sequencing data for 19 neuroblastoma trios each including constitutional, diagnosis and relapse samples. We confirmed an enrichment of damaging variants within such pathways as MAPK (mitogen-activated protein kinases), neuritogenesis, epithelial-mesenchymal transition, cell survival and DNA repair. Most pathways had more damaging variants in the expanding clones compared to shrinking ones, which can be explained by the increased total number of variants between these two populations. Functional mutational rate varied for ancestral clones and clones shrinking or expanding upon treatment, suggesting changes in clone selection mechanisms at different time points of tumor evolution. Availability and implementation: Source code and binaries of the QuantumClone R package are freely available for download at https://CRAN.R-project.org/package=QuantumClone. Contact: gudrun.schleiermacher@curie.fr or valentina.boeva@inserm.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

ERBB3-Activating Mutations in Small Bowel Adenocarcinomas.

PURPOSE: Functional studies have demonstrated that some mutations of ERBB3, which encodes for human epidermal growth factor receptor (HER) 3, are oncogenic via activation of the ErbB family signaling pathway. Significant clinical activity of anti-HER2 therapies (trastuzumab plus lapatinib combination or afatinib) has been reported in patients with ERBB3-mutated cancers. This study was designed to report the rate of activating ERBB3 mutations in small bowel adenocarcinoma (SBA), a rare tumor type in which we previously reported a high rate (12%) of ERBB2-activating mutations. MATERIALS AND METHODS: DNA from 74 SBAs, previously characterized for ERBB2 mutations and mismatch repair status, was submitted for sequencing of ERBB3 exons 3, 6, 7, 8, and 23. Orthogonal validation by targeted next-generation sequencing was performed. RESULTS: Four of 74 SBAs (5.4%) displayed ERBB3-activating mutations, including three p.V104M mutations (c.310 G>A) in exon 3 and one p.E928G mutation (c.2783 A>G) in exon 23. No mutations were detected in exons 6, 7, and 8. ERBB3-activating mutations were associated with microsatellite instability (P = .002) and the presence of ERBB2-activating mutations (P = .002). Two SBAs with co-occurrence of ERBB2 and ERBB3 mutations were further analyzed by targeted next-generation sequencing. Mutant allelic frequencies suggested that both mutations were shared by the same clone rather than being harbored by mutually exclusive tumor subclones. CONCLUSION: SBAs display a high rate of ERBB3-activating mutations, which have been shown to be targetable by anti-HER2 therapies. Strikingly, ERBB3 was frequently comutated with ERBB2, suggesting a strong oncogenic addiction of these SBAs to the HER2 pathway.

Whole-Exome Sequencing of Cell-Free DNA Reveals Temporo-spatial Heterogeneity and Identifies Treatment-Resistant Clones in Neuroblastoma.

Purpose: Neuroblastoma displays important clinical and genetic heterogeneity, with emergence of new mutations at tumor progression.Experimental Design: To study clonal evolution during treatment and follow-up, an innovative method based on circulating cell-free DNA (cfDNA) analysis by whole-exome sequencing (WES) paired with target sequencing was realized in sequential liquid biopsy samples of 19 neuroblastoma patients.Results: WES of the primary tumor and cfDNA at diagnosis showed overlap of single-nucleotide variants (SNV) and copy number alterations, with 41% and 93% of all detected alterations common to the primary neuroblastoma and cfDNA. CfDNA WES at a second time point indicated a mean of 22 new SNVs for patients with progressive disease. Relapse-specific alterations included genes of the MAPK pathway and targeted the protein kinase A signaling pathway. Deep coverage target sequencing of intermediate time points during treatment and follow-up identified distinct subclones. For 17 seemingly relapse-specific SNVs detected by cfDNA WES at relapse but not tumor or cfDNA WES at diagnosis, deep coverage target sequencing detected these alterations in minor subclones, with relapse-emerging SNVs targeting genes of neuritogenesis and cell cycle. Furthermore a persisting, resistant clone with concomitant disappearance of other clones was identified by a mutation in the ubiquitin protein ligase HERC2Conclusions: Modelization of mutated allele fractions in cfDNA indicated distinct patterns of clonal evolution, with either a minor, treatment-resistant clone expanding to a major clone at relapse, or minor clones collaborating toward tumor progression. Identification of treatment-resistant clones will enable development of more efficient treatment strategies. Clin Cancer Res; 24(4); 939-49. ©2017 AACR.

Feasibility and clinical integration of molecular profiling for target identification in pediatric solid tumors.

BACKGROUND: The role of tumor molecular profiling in directing targeted therapy utilization remains to be defined for pediatric tumors. We aimed to evaluate the feasibility of a sequencing and molecular biology tumor board (MBB) program, and its clinical impact on children with solid tumors. PROCEDURE: We report on a single-center MBB experience of 60 pediatric patients with a poor prognosis or relapsed/refractory solid tumors screened between October 2014 and November 2015. Tumor molecular profiling was performed with panel-based next-generation sequencing and array comparative genomic hybridization. RESULTS: Mean age was 12 ± 5.7 years (range 0.1-21.5); main tumor types were high-grade gliomas (n = 14), rare sarcomas (n = 9), and neuroblastomas (n = 8). The indication was a poor prognosis tumor at diagnosis for 16 patients and relapsed (n = 26) or refractory disease (n = 18) for the remaining 44 patients. Molecular profiling was feasible in 58 patients. Twenty-three patients (40%) had a potentially actionable finding. Patients with high-grade gliomas had the highest number of targetable alterations (57%). Six of the 23 patients subsequently received a matched targeted therapy for a period ranging from 16 days to 11 months. The main reasons for not receiving targeted therapy were poor general condition (n = 5), pursuit of conventional therapy (n = 6), or lack of pediatric trial (n = 4). CONCLUSIONS: Pediatric molecular profiling is feasible, with more than a third of patients being eligible to receive targeted therapy, yet only a small proportion were treated with these therapies. Analysis at diagnosis may be useful for children with very poor prognosis tumsors.

Identification of non-coding genetic variants in samples from hypoxemic respiratory disease patients that affect the transcriptional response to hypoxia.

A wide range of diseases course with an unbalance between the consumption of oxygen by tissues and its supply. This situation triggers a transcriptional response, mediated by the hypoxia inducible factors (HIFs), that aims to restore oxygen homeostasis. Little is known about the inter-individual variation in this response and its role in the progression of disease. Herein, we sought to identify common genetic variants mapping to hypoxia response elements (HREs) and characterize their effect on transcription. To this end, we constructed a list of genome-wide HIF-binding regions from publicly available experimental datasets and studied the genetic variability in these regions by targeted re-sequencing of genomic samples from 96 chronic obstructive pulmonary disease and 144 obstructive sleep apnea patients. This study identified 14 frequent variants disrupting potential HREs. The analysis of the genomic regions containing these variants by means of reporter assays revealed that variants rs1009329, rs6593210 and rs150921338 impaired the transcriptional response to hypoxia. Finally, using genome editing we confirmed the functional role of rs6593210 in the transcriptional regulation of EGFR. In summary, we found that inter-individual variability in non-coding regions affect the response to hypoxia and could potentially impact on the progression of pulmonary diseases.

NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.

Genomic Copy Number Profiling Using Circulating Free Tumor DNA Highlights Heterogeneity in Neuroblastoma.

PURPOSE: The tumor genomic copy number profile is of prognostic significance in neuroblastoma patients. We have studied the genomic copy number profile of cell-free DNA (cfDNA) and compared this with primary tumor arrayCGH (aCGH) at diagnosis. EXPERIMENTAL DESIGN: In 70 patients, cfDNA genomic copy number profiling was performed using the OncoScan platform. The profiles were classified according to the overall pattern, including numerical chromosome alterations (NCA), segmental chromosome alterations (SCA), and MYCN amplification (MNA). RESULTS: Interpretable and dynamic cfDNA profiles were obtained in 66 of 70 and 52 of 70 cases, respectively. An overall identical genomic profile between tumor aCGH and cfDNA was observed in 47 cases (3 NCAs, 22 SCAs, 22 MNAs). In one case, cfDNA showed an additional SCA not detected by tumor aCGH. In 4 of 8 cases with a silent tumor aCGH profile, cfDNA analysis revealed a dynamic profile (3 SCAs, 1 NCA). In 14 cases, cfDNA analysis did not reveal any copy number changes. A total of 378 breakpoints common to the primary tumor and cfDNA of any given patient were identified, 27 breakpoints were seen by tumor aCGH, and 54 breakpoints were seen in cfDNA only, including two cases with interstitial IGFR1 gains and two alterations targeting TERT CONCLUSIONS: These results demonstrate the feasibility of cfDNA copy number profiling in neuroblastoma patients, with a concordance of the overall genomic profile in aCGH and cfDNA dynamic cases of 97% and a sensitivity of 77%, respectively. Furthermore, neuroblastoma heterogeneity is highlighted, suggesting that cfDNA might reflect genetic alterations of more aggressive cell clones. Clin Cancer Res; 22(22); 5564-73. ©2016 AACRSee related commentary by Janku and Kurzrock, p. 5400.

Frequent Somatic Mutation in Adult Intestinal Stem Cells Drives Neoplasia and Genetic Mosaicism during Aging.

Adult stem cells may acquire mutations that modify cellular behavior, leading to functional declines in homeostasis or providing a competitive advantage resulting in premalignancy. However, the frequency, phenotypic impact, and mechanisms underlying spontaneous mutagenesis during aging are unclear. Here, we report two mechanisms of genome instability in adult Drosophila intestinal stem cells (ISCs) that cause phenotypic alterations in the aging intestine. First, we found frequent loss of heterozygosity arising from mitotic homologous recombination in ISCs that results in genetic mosaicism. Second, somatic deletion of DNA sequences and large structural rearrangements, resembling those described in cancers and congenital diseases, frequently result in gene inactivation. Such modifications induced somatic inactivation of the X-linked tumor suppressor Notch in ISCs, leading to spontaneous neoplasias in wild-type males. Together, our findings reveal frequent genomic modification in adult stem cells and show that somatic genetic mosaicism has important functional consequences on aging tissues.

Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, open-label, proof-of-concept, randomised, controlled phase 2 trial.

BACKGROUND: Molecularly targeted agents have been reported to have anti-tumour activity for patients whose tumours harbour the matching molecular alteration. These results have led to increased off-label use of molecularly targeted agents on the basis of identified molecular alterations. We assessed the efficacy of several molecularly targeted agents marketed in France, which were chosen on the basis of tumour molecular profiling but used outside their indications, in patients with advanced cancer for whom standard-of-care therapy had failed. METHODS: The open-label, randomised, controlled phase 2 SHIVA trial was done at eight French academic centres. We included adult patients with any kind of metastatic solid tumour refractory to standard of care, provided they had an Eastern Cooperative Oncology Group performance status of 0 or 1, disease that was accessible for a biopsy or resection of a metastatic site, and at least one measurable lesion. The molecular profile of each patient's tumour was established with a mandatory biopsy of a metastatic tumour and large-scale genomic testing. We only included patients for whom a molecular alteration was identified within one of three molecular pathways (hormone receptor, PI3K/AKT/mTOR, RAF/MEK), which could be matched to one of ten regimens including 11 available molecularly targeted agents (erlotinib, lapatinib plus trastuzumab, sorafenib, imatinib, dasatinib, vemurafenib, everolimus, abiraterone, letrozole, tamoxifen). We randomly assigned these patients (1:1) to receive a matched molecularly targeted agent (experimental group) or treatment at physician's choice (control group) by central block randomisation (blocks of size six). Randomisation was done centrally with a web-based response system and was stratified according to the Royal Marsden Hospital prognostic score (0 or 1 vs 2 or 3) and the altered molecular pathway. Clinicians and patients were not masked to treatment allocation. Treatments in both groups were given in accordance with the approved product information and standard practice protocols at each institution and were continued until evidence of disease progression. The primary endpoint was progression-free survival in the intention-to-treat population, which was not assessed by independent central review. We assessed safety in any patients who received at least one dose of their assigned treatment. This trial is registered with ClinicalTrials.gov, number NCT01771458. FINDINGS: Between Oct 4, 2012, and July 11, 2014, we screened 741 patients with any tumour type. 293 (40%) patients had at least one molecular alteration matching one of the 10 available regimens. At the time of data cutoff, Jan 20, 2015, 195 (26%) patients had been randomly assigned, with 99 in the experimental group and 96 in the control group. All patients in the experimental group started treatment, as did 92 in the control group. Two patients in the control group received a molecularly targeted agent: both were included in their assigned group for efficacy analyses, the patient who received an agent that was allowed in the experimental group was included in the experimental group for the purposes of safety analyses, while the other patient, who received a molecularly targeted agent and chemotherapy, was kept in the control group for safety analyses. Median follow-up was 11·3 months (IQR 5·8-11·6) in the experimental group and 11·3 months (8·1-11·6) in the control group at the time of the primary analysis of progression-free survival. Median progression-free survival was 2·3 months (95% CI 1·7-3·8) in the experimental group versus 2·0 months (1·8-2·1) in the control group (hazard ratio 0·88, 95% CI 0·65-1·19, p=0·41). In the safety population, 43 (43%) of 100 patients treated with a molecularly targeted agent and 32 (35%) of 91 patients treated with cytotoxic chemotherapy had grade 3-4 adverse events (p=0·30). INTERPRETATION: The use of molecularly targeted agents outside their indications does not improve progression-free survival compared with treatment at physician's choice in heavily pretreated patients with cancer. Off-label use of molecularly targeted agents should be discouraged, but enrolment in clinical trials should be encouraged to assess predictive biomarkers of efficacy.