Functional characterization of 5p15.33 risk locus in uveal melanoma reveals rs452384 as a functional variant and NKX2.4 as an allele-specific interactor.

The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.

Mosaic BRCA1 promoter methylation contribution in hereditary breast/ovarian cancer pedigrees.

PURPOSE: Mosaic BRCA1 promoter methylation (BRCA1meth) increases the risk of early-onset breast cancer, triple-negative breast cancer and ovarian cancer. As mosaic BRCA1meth are believed to occur de novo, their role in family breast/ovarian cancer has not been assessed. PATIENTS: Blood-derived DNA from 20 unrelated affected cases from families with aggregation of breast/ovarian cancer, but with no germline pathogenic variants in BRCA1/2, PALB2 or RAD51C/D, were screened by methylation-sensitive high-resolution melting. CpG analysis was performed by pyrosequencing on blood and buccal swab. Two probands carried a pathogenic variant in a moderate-penetrance gene (ATM and BARD1), and 8 of 18 others (44%) carried BRCA1meth (vs none of the 20 age-matched controls). Involvement of BRCA1 in tumourigenesis in methylated probands was demonstrated in most tested cases by detection of a loss of heterozygosity and a homologous recombination deficiency signature. Among the eight methylated probands, two had relatives with breast cancer with detectable BRCA1meth in blood, including one with high methylation levels in two non-tumour tissues. CONCLUSIONS: The high prevalence of mosaic BRCA1meth in patients with breast/ovarian cancer with affected relatives, as well as this first description of a family aggregation of mosaic BRCA1meth, shows how this de novo event can contribute to hereditary breast/ovarian cancer pedigrees.

Importance of pseudoaneurysms after TAVI - a retrospective analysis of 2063 patients.

Background: Bifemoral arterial access is common in patients undergoing transcatheter aortic valve implantation (TAVI), with a primary treatment access (TAVI access) and a secondary non-TAVI access. Pseudoaneurysm (PSA) is an important complication of femoral arterial puncture. Major vascular complications after TAVI are well described, but little is known about PSA. Patients and methods: A total of 2063 patients underwent transfemoral TAVI between January 2014 and January 2020. Vascular ultrasound of the common femoral artery was assessed before and after TAVI. We compared patient characteristics, periprocedural risk scores, procedural characteristics, and access site bleeding events according to Valve Academic Research Consortium 3 (VARC-3) criteria, length of stay (LOS), and all-cause mortality at one year between patients with (46) and without (2017) PSA. Results: The incidence of PSA after TAVI was 2.2% (46/2063). All PSA were successfully treated with ultrasound-guided manual compression (UGMC) or thrombin injection (UGTI) without complications. Patients with PSA had lower platelet counts (210×1000/µl vs. 234×1000/µl; p<0.05), more heart failure symptoms on admission (91% vs. 25%; p<0.05), were more often treated with (N)OACs for atrial fibrillation (AF; 54% vs. 38%; p <0.05), and were less often treated with aspirin (35% vs. 51%; p<0.03). Multivariate analysis identified secondary access site (odds ratio [OR] 8.11; p<0.001) and (N)OAC therapy (OR 1.31; p = 0.037) as risk factors for PSA development. PSA is associated with VARC-3 type 1-3 access site bleeding and longer LOS (15.2 ± 11.3 d vs. 11.6 ± 8.9 d; p<0.01), but this did not affect one year mortality (17% vs. 14%; p = 0.53). Conclusions: Pseudoaneurysms are an important complication after TAVI and are associated with access site bleeding and prolonged hospital stay. (N)OAC therapy and secondary access are important risk factors. Pseudoaneurysms can be safely and effectively treated with thrombin injection and do not affect one-year mortality.

Immunohistochemical characterisation of the immune landscape in primary uveal melanoma and liver metastases.

BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.

Circulating Tumor DNA as a Prognostic Factor in Patients With Resectable Hepatic Metastases of Uveal Melanoma.

OBJECTIVE: We report here the results of a prospective study of circulating tumor DNA (ctDNA) detection in patients undergoing uveal melanoma (UM) liver metastases resection (NCT02849145). BACKGROUND: In UM patients, the liver is the most common and often only site of metastases. Local treatments of liver metastases, such as surgical resection, have a likely benefit in selected patients. METHODS: Upon enrollment, metastatic UM patients eligible for curative liver surgery had plasma samples collected before and after surgery. GNAQ / GNA11 mutations were identified in archived tumor tissue and used to quantify ctDNA by droplet digital polymerase chain reaction which was then associated with the patient's surgical outcomes. RESULTS: Forty-seven patients were included. Liver surgery was associated with a major increase of cell-free circulating DNA levels, with a peak 2 days after surgery (∼20-fold). Among 40 evaluable patients, 14 (35%) had detectable ctDNA before surgery, with a median allelic frequency of 1.1%. These patients experienced statistically shorter relapse-free survival (RFS) versus patients with no detectable ctDNA before surgery (median RFS: 5.5 vs 12.2 months; hazard ratio=2.23, 95% CI: 1.06-4.69, P =0.04), and had a numerically shorter overall survival (OS) (median OS: 27.0 vs 42.3 months). ctDNA positivity at postsurgery time points was also associated with RFS and OS. CONCLUSIONS: This study is the first to report ctDNA detection rate and prognostic impact in UM patients eligible for surgical resection of their liver metastases. If confirmed by further studies in this setting, this noninvasive biomarker could inform treatment decisions in UM patients with liver metastases.

Results of Lung Transplantation for Cystic Fibrosis With Selected Donors Over 65 Years Old.

Lung transplantation is limited by the shortage of suitable donors. Many programs have begun to use extended criteria donors. Donors over 65 years old are rarely reported, especially for young cystic fibrosis recipients. This monocentric study was conducted for cystic fibrosis recipients from January 2005 to December 2019, comparing two cohorts according to lung donor age (<65 years or ≥65 years). The primary objective was to assess the survival rate at 3 years using a Cox multivariable model. Of the 356 lung recipients, 326 had donors under 65 years, and 30 had donors over 65 years. Donors' characteristics did not differ significantly in terms of sex, time on mechanical ventilation before retrieval, and partial pressure of arterial oxygen/fraction of inspired oxygen ratio. There were no significant differences in post-operative mechanical ventilation duration and incidence of grade 3 primary graft dysfunction between the two groups. At 1, 3, and 5 years, the percentage of predicted forced expiratory volume in 1 s (p = 0.767) and survival rate did not differ between groups (p = 0.924). The use of lungs from donors over 65 years for cystic fibrosis recipients allows extension of the donor pool without compromising results. Longer follow-up is needed to assess the long-term effects of this practice.

Real-Time Detection of ESR1 Mutation in Blood by Droplet Digital PCR in the PADA-1 Trial: Feasibility and Cross-Validation with NGS.

The clinical actionability of circulating tumor DNA requires sensitive detection methods with a short turnaround time. In the PADA-1 phase 3 trial (NCT03079011), metastatic breast cancer patients treated with an aromatase inhibitor and palbociclib were screened every 2 months for activating ESR1 mutations in blood (bESR1mut). We report the feasibility of the droplet digital polymerase chain reaction (ddPCR) and cross-validation with next-generation sequencing (NGS). bESR1mut testing was centralized in two platforms using the same ddPCR assay. Results were reported as copies/mL of plasma and mutant allele frequency (MAF). We analyzed 200 positive ddPCR samples with an NGS assay (0.5-1% sensitivity). Overall, 12,552 blood samples were collected from 1017 patients from 83 centers. Among the 12,525 available samples with ddPCR results, 11,533 (92%) were bESR1mut-negative. A total of 267 patients newly displayed bESR1mut (26% patients/2% samples) with a median copy number of 14/mL (range: 4-1225) and a median MAF of 0.83% (0.11-35), 648 samples (20% patients/5% samples) displayed persistent bESR1mut, and 77 (<1%) samples encountered a technical failure. The median turnaround time from blood drawing to result notification was 13 days (Q1:9; Q3:21 days). Among 200 ddPCR-positive samples tested, NGS detected bESR1mut in 168 (84%); 25 of the 32 cases missed by NGS had low MAF and/or low coverage. In these 200 samples, bESR1mut MAF by both techniques had an excellent intraclass correlation coefficient (ICC = 0.93; 95% CI [0.85; 0.97]). These results from a large-scale trial support the feasibility and accuracy of real-time bESR1mut tracking by ddPCR, opening new opportunities for therapeutic interventions.

Chromosome 3 and 8q Aberrations in Uveal Melanoma Show Greater Impact on Survival in Patients with Light Iris versus Dark Iris Color.

PURPOSE: Individuals with gray, blue, or green eyes have a higher chance of developing uveal melanoma (UM) than those with brown eyes. We wondered whether iris pigmentation might be related not only to predisposition to UM but also to its behavior; therefore, we compared the clinical, histopathologic, and genetic characteristics of UM between eyes with different colors. DESIGN: We determined iris color in a large cohort of patients enucleated for UM. Clinical and histopathologic tumor characteristics, chromosome status, and survival were compared among 3 groups on the basis of iris color. PARTICIPANTS: A total of 412 patients with choroidal/ciliary body UM, who had undergone primary enucleation at the Leiden University Medical Center, Leiden, The Netherlands, between 1993 and 2019, were divided into 3 groups based on iris color: gray/blue, green/hazel, and brown. The validation cohort included 934 patients with choroidal/ciliary body UM treated at Wills Eye Hospital (WEH). METHODS: Comparison of clinical, histopathologic, and genetic characteristics of UM in patients with different iris colors. MAIN OUTCOME MEASURES: Melanoma-related survival in UM patients, divided over 3 iris color groups, in relation to the tumor's chromosome 3 and 8q status. RESULTS: Moderate and heavy tumor pigmentations were especially seen in eyes with a brown iris (P < 0.001). Survival did not differ between patients with different iris colors (P = 0.27); however, in patients with a light iris, copy number changes in chromosome 3 and 8q had a greater influence on survival than in patients with a dark iris. Likewise, chromosome 3 and chromosome 8q status affected survival more among patients with lightly pigmented tumors than in patients with heavily pigmented tumors. The WEH cohort similarly showed a greater influence of chromosome aberrations in light-eyed individuals. CONCLUSIONS: Although iris color by itself did not relate to UM-related survival, chromosome 3 and 8q aberrations had a larger influence on survival in patients with a light iris than those with a brown iris. This suggests a synergistic effect of iris pigmentation and chromosome status in the regulation of oncogenic behavior of UM. Iris color should be taken into consideration when calculating a patient's risk for developing metastases.

Acquired platelet storage container leaks and contamination with environmental bacteria: A preventable cause of bacterial sepsis.

BACKGROUND: Apheresis platelets (AP) may be contaminated by environmental bacteria via container defects acquired during processing, transport, storage, or transfusion, as highlighted by a recent series of septic reactions related to Acinetobacter spp. and other bacterial strains. STUDY DESIGN AND METHODS: The frequency and nature of acquired container defect reports to one manufacturer were evaluated from January 2019 to July 2020. The published incidence of contamination and sepsis due to environmental bacteria with culture screened AP in the United States was reviewed for the period of 2010-2019. RESULTS: Review of a manufacturers' records showed 23 US reports of leaks involving 24 containers attributed to postmanufacturing damage, at a rate of 44 per million distributed storage containers. Analysis of returned containers showed evidence of scratches, impressions, and/or piercings. Literature review of US hemovigilance data revealed that environmental bacteria comprised 7% of confirmed positive primary bacterial culture screens, were responsible for 14%-16% of reported septic, and 8 of 28 (29%) fatal reactions with bacterial-culture screened AP. Sepsis cases have been reported with culture screened, point-of-issue (POI) tested, or pathogen-reduced AP. DISCUSSION: Environmental contamination of AP is rare but can cause sepsis. Container damage provides a pathway for contamination after culture screening, POI bacteria testing, or pathogen reduction. Blood collectors and transfusion services should have procedures to ensure proper inspection, handling, storage, and transport of AP to avoid damage and should enhance efforts to detect defects prior to release and to eliminate bacteria from all contacting surfaces to minimize the risk of contamination.

ShallowHRD: detection of homologous recombination deficiency from shallow whole genome sequencing.

SUMMARY: We introduce shallowHRD, a software tool to evaluate tumor homologous recombination deficiency (HRD) based on whole genome sequencing (WGS) at low coverage (shallow WGS or sWGS; ∼1X coverage). The tool, based on mining copy number alterations profile, implements a fast and straightforward procedure that shows 87.5% sensitivity and 90.5% specificity for HRD detection. shallowHRD could be instrumental in predicting response to poly(ADP-ribose) polymerase inhibitors, to which HRD tumors are selectively sensitive. shallowHRD displays efficiency comparable to most state-of-art approaches, is cost-effective, generates low-storable outputs and is also suitable for fixed-formalin paraffin embedded tissues. AVAILABILITY AND IMPLEMENTATION: shallowHRD R script and documentation are available at https://github.com/aeeckhou/shallowHRD. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteomic evidence of specific IGKV1-8 association with cystic lung light chain deposition disease.

We previously reported a new form of light chain deposition disease (LCDD) presenting as diffuse cystic lung disorder that differs from the usual systemic form with respect to patient age, the male/female ratio, the involved organs, and the hematologic characteristics. We also demonstrated that the light chains were produced by an intrapulmonary B-cell clone and that this clone shared a stereotyped antigen receptor IGHV4-34/IGKV1. However, we only analyzed 3 patients. We conducted a retrospective study including lung tissue samples from 24 patients with pulmonary LCDD (pLCDD) matched with samples from 13 patients with pulmonary κ light chain amyloidosis (pAL amyloidosis) used as controls. Mass spectrometry-based proteomics identified immunoglobulin κ peptides as the main protein component of the tissue deposits in all patients. Interestingly, in pLCDD, IGKV1 was the most common κ family detected (86.4%), and IGKV1-8 was overrepresented compared with pAL amyloidosis (75% vs 11.1%, P = .0033). Furthermore, IGKV1-8 was predominantly associated with a diffuse cystic pattern (94%) in pLCDD. In conclusion, the high frequency of IGKV1-8 usage in cystic pLCDD constitutes an additional feature arguing for a specific entity distinct from the systemic form that preferentially uses IGKV4-1.

Willingness to Receive a COVID-19 Vaccination Among Incarcerated or Detained Persons in Correctional and Detention Facilities - Four States, September-December 2020.

Incarcerated and detained persons are at increased risk for acquiring COVID-19. However, little is known about their willingness to receive a COVID-19 vaccination. During September-December 2020, residents in three prisons and 13 jails in four states were surveyed regarding their willingness to receive a COVID-19 vaccination and their reasons for COVID-19 vaccination hesitancy or refusal. Among 5,110 participants, 2,294 (44.9%) said they would receive a COVID-19 vaccination, 498 (9.8%) said they would hesitate to receive it, and 2,318 (45.4%) said they would refuse to receive it. Willingness to receive a COVID-19 vaccination was lowest among Black/African American (Black) (36.7%; 510 of 1,390) persons, participants aged 18-29 years (38.5%; 583 of 1,516), and those who lived in jails versus prisons (43.7%; 1,850 of 4,232). Common reasons reported for COVID-19 vaccine hesitancy were waiting for more information (54.8%) and efficacy or safety concerns (31.0%). The most common reason for COVID-19 vaccination refusal was distrust of health care, correctional, or government personnel or institutions (20.1%). Public health interventions to improve vaccine confidence and trust are needed to increase vaccination acceptance by incarcerated or detained persons.

DNA repair functional analyses of NBN hypomorphic variants associated with NBN-related infertility.

Nijmegen breakage syndrome caused by biallelic pathogenic variants of the DNA-damage response gene NBN, is characterized by severe microcephaly, cancer proneness, infertility, and karyotype abnormalities. We previously reported NBN variants in siblings suffering from fertility defects. Here, we identify a new founder NBN variant (c.442A>G, p.(Thr148Ala)) in Lebanese patients associated with isolated infertility. Functional analyses explored preserved or altered functions correlated with their remarkably mild phenotype. Transcript and protein analyses supported the use of an alternative transcript with in-frame skipping of exons 4-5, leading to p84-NBN protein with a preserved forkhead-associated (FHA) domain. The level of NBN was dramatically reduced and the MRN complex delocalized to the cytoplasm. Interestingly, ataxia-elangiectasia mutated (ATM) also shifted from the nucleus to the cytoplasm, suggesting some interaction between ATM and the MRN complex at a steady state. The ATM pathway activation, attenuated in typical patients with NBS, appeared normal under camptothecin treatment in these new NBN-related infertile patients. Cell cycle checkpoint defect was present in these atypical patients, although to a lesser extent than in typical patients with NBS. In conclusion, we report three new NBN-related infertile patients and we suggest that preserved FHA domain could be responsible for the mild phenotype and intermediate DNA-damage response defects.

High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies.

BACKGROUND: Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. METHODS: We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. RESULTS: The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. CONCLUSIONS: This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

Addressing identity-related barriers to collaboration for conservation through self-affirmation theory and moral foundations theory.

The natural resource management literature documents many reasons for pursuing collaborative processes, offering useful insights on how to manage conflict and facilitate productive deliberation in complex multistakeholder collaborative efforts. Moral foundations theory and self-affirmation theory can further help collaborative efforts mitigate conflicts caused by identity threats and the identity-protective reasoning these threats provoke. Moral foundations theory suggests an approach to increase collaboration by minimizing triggering language and helping people appreciate opposing viewpoints. Self-affirmation theory suggests a practical intervention that could be used to increase collaboration by desensitizing people to identity threats and reducing defensiveness. Taken together, these theories can contribute substantially to the understanding and practice of collaboration and conflict management for conservation.

Consideración de las Barreras Relacionadas con la Identidad que Enfrenta la Colaboración para la Conservación a través de la Teoría de Autoafirmación y la Teoría de Fundamentos Morales Resumen La literatura sobre el manejo de recursos naturales documenta muchas razones por las que es necesario buscar procesos colaborativos, los cuales ofrecen conocimiento útil sobre cómo manejar el conflicto. Estos procesos también facilitan la deliberación productiva dentro de los esfuerzos colaborativos complejos en los cuales participan múltiples actores. La teoría de fundamentos morales puede ayudar a que los esfuerzos colaborativos mitiguen los conflictos causados por las amenazas a la identidad y el razonamiento de protección de identidad que estas amenazas provocan. La teoría de fundamentos morales propone una estrategia para incrementar la colaboración al minimizar el lenguaje detonante y ayudar a que las personas aprecien los puntos de vista contrarios. La teoría de autoafirmación sugiere una intervención práctica que podría usarse para incrementar la colaboración al desensibilizar a las personas a tal grado que identifiquen amenazas y reduzcan la actitud defensiva. En conjunto, estas teorías pueden contribuir sustancialmente al entendimiento y la práctica de la colaboración y el manejo de conflictos para la conservación.

Three new cases of ataxia-telangiectasia-like disorder: No impairment of the ATM pathway, but S-phase checkpoint defect.

Ataxia-telangiectasia-like disorder (ATLD) is a rare genomic instability syndrome caused by biallelic variants of MRE11 (meiotic recombination 11) characterized by progressive cerebellar ataxia and typical karyotype abnormalities. These symptoms are common to those of ataxia-telangiectasia, which is consistent with the key role of MRE11 in ataxia-telangiectasia mutated (ATM) activation after DNA double-strand breaks. Three unrelated French patients were referred with ataxia. Only one had typical karyotype abnormalities. Unreported biallelic MRE11 variants were found in these three cases. Interestingly, one variant (c.424G>A) was present in two cases and haplotype analysis strongly suggested a French founder variant. Variants c.544G>A and c.314+4_314+7del lead to splice defects. The level of MRE11 in lymphoblastoid cell lines was consistently and dramatically reduced. Functional consequences were evaluated on activation of the ATM pathway via phosphorylation of ATM targets (KAP1 and CHK2), but no consistent defect was observed. However, an S-phase checkpoint activation defect after camptothecin was observed in these patients with ATLD. In conclusion, we report the first three French ATLD patients and a French founder variant, and propose an S-phase checkpoint activation study to evaluate the pathogenicity of MRE11 variants.

Functional classification of ATM variants in ataxia-telangiectasia patients.

Ataxia-telangiectasia (A-T) is a recessive disorder caused by biallelic pathogenic variants of ataxia-telangiectasia mutated (ATM). This disease is characterized by progressive ataxia, telangiectasia, immune deficiency, predisposition to malignancies, and radiosensitivity. However, hypomorphic variants may be discovered associated with very atypical phenotypes, raising the importance of evaluating their pathogenic effects. In this study, multiple functional analyses were performed on lymphoblastoid cell lines from 36 patients, comprising 49 ATM variants, 24 being of uncertain significance. Thirteen patients with atypical phenotype and presumably hypomorphic variants were of particular interest to test strength of functional analyses and to highlight discrepancies with typical patients. Western-blot combined with transcript analyses allowed the identification of one missing variant, confirmed suspected splice defects and revealed unsuspected minor transcripts. Subcellular localization analyses confirmed the low level and abnormal cytoplasmic localization of ATM for most A-T cell lines. Interestingly, atypical patients had lower kinase defect and less altered cell-cycle distribution after genotoxic stress than typical patients. In conclusion, this study demonstrated the pathogenic effects of the 49 variants, highlighted the strength of KAP1 phosphorylation test for pathogenicity assessment and allowed the establishment of the Ataxia-TeLangiectasia Atypical Score to predict atypical phenotype. Altogether, we propose strategies for ATM variant detection and classification.

Medullary Breast Carcinoma, a Triple-Negative Breast Cancer Associated with BCLG Overexpression.

Medullary breast carcinoma (MBC) is a rare subtype of triple-negative breast cancer with specific genomic features within the spectrum of basal-like carcinoma (BLC). In this study of 19 MBCs and 36 non-MBC BLCs, we refined the transcriptomic and genomic knowledge about this entity. Unsupervised and supervised analysis of transcriptomic profiles confirmed that MBC clearly differs from non-MBC BLC, with 92 genes overexpressed and 154 genes underexpressed in MBC compared with non-MBC BLC. Immunity-related pathways are the most differentially represented pathways in MBC compared with non-MBC BLC. The proapoptotic gene BCLG (official name BCL2L14) is by far the most intensely overexpressed gene in MBC. A quantitative RT-PCR validation study conducted in 526 breast tumors corresponding to all molecular subtypes documented the specificity of BCLG overexpression in MBC, which was confirmed at the protein level by immunohistochemistry. We also found that most MBCs belong to the immunomodulatory triple-negative breast cancer subtype. Using pan-genomic analysis, it was found that MBC harbors more losses of heterozygosity than non-MBC BLC. These observations corroborate the notion that MBC remains a distinct entity that could benefit from specific treatment strategies (such as deescalation or targeted therapy) adapted to this rare tumor type.

The inactive X chromosome is epigenetically unstable and transcriptionally labile in breast cancer.

Disappearance of the Barr body is considered a hallmark of cancer, although whether this corresponds to genetic loss or to epigenetic instability and transcriptional reactivation is unclear. Here we show that breast tumors and cell lines frequently display major epigenetic instability of the inactive X chromosome, with highly abnormal 3D nuclear organization and global perturbations of heterochromatin, including gain of euchromatic marks and aberrant distributions of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells and a significant degree of aberrant gene activity from the inactive X chromosome, including several genes involved in cancer promotion. We demonstrate that many of these genes are aberrantly reactivated in primary breast tumors, and we further demonstrate that epigenetic instability of the inactive X can lead to perturbed dosage of X-linked factors. Taken together, our study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the inactive X can lead to the disappearance of the Barr body in breast cancer cells. This work offers new insights and opens up the possibility of exploiting the inactive X chromosome as an epigenetic biomarker at the molecular and cytological levels in cancer.

Lung transplantation after allogeneic stem cell transplantation: a pan-European experience.

Late-onset noninfectious pulmonary complications (LONIPCs) affect 6% of allogeneic stem cell transplantation (SCT) recipients within 5 years, conferring subsequent 5-year survival of 50%. Lung transplantation is rarely performed in this setting due to concomitant extrapulmonary morbidity, excessive immunosuppression and concerns about recurring malignancy being considered contraindications. This study assesses survival in highly selected patients undergoing lung transplantation for LONIPCs after SCT.SCT patients undergoing lung transplantation at 20 European centres between 1996 and 2014 were included. Clinical data pre- and post-lung transplantation were reviewed. Propensity score-matched controls were generated from the Eurotransplant and Scandiatransplant registries. Kaplan-Meier survival analysis and Cox proportional hazard regression models evaluating predictors of graft loss were performed.Graft survival at 1, 3 and 5 years of 84%, 72% and 67%, respectively, among the 105 SCT patients proved comparable to controls (p=0.75). Sepsis accounted for 15 out of 37 deaths (41%), with prior mechanical ventilation (HR 6.9, 95% CI 1.0-46.7; p<0.001) the leading risk factor. No SCT-specific risk factors were identified. Recurring malignancy occurred in four patients (4%). Lung transplantation <2 years post-SCT increased all-cause 1-year mortality (HR 7.5, 95% CI 2.3-23.8; p=0.001).Lung transplantation outcomes following SCT were comparable to other end-stage diseases. Lung transplantation should be considered feasible in selected candidates. No SCT-specific factors influencing outcome were identified within this carefully selected patient cohort.