The Role of Fragile Sites in Sporadic Papillary Thyroid Carcinoma

The incidence of thyroid cancer is increasing, especially papillary thyroid carcinoma (PTC), making it currently the fastest-growing cancer among women. Reasons for this increase remain unclear, but several risk factors including radiation exposure and improved detection techniques have been suggested. Recently, the induction of chromosomal fragile site breakage was found to result in the formation of RET/PTC1 rearrangements, a common cause of PTC. Chromosomal fragile sites are regions of the genome with a high susceptibility to forming DNA breaks and are often associated with cancer. Exposure to a variety of external agents can induce fragile site breakage, which may account for some of the observed increase in PTC. This paper discusses the role of fragile site breakage in PTC development, external fragile site-inducing agents that may be potential risk factors for PTC, and how these factors are especially targeting women

DNA Instability at Chromosomal Fragile Sites in Cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development

Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity

SummaryMicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density from promoters with activating chromatin modifications and in sperm from the 5′-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveals that homologous recombination and non-homologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology—either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair

DNA topoisomerases participate in fragility of the oncogene RET

Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APHinduced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication

Allogeneic Hematopoietic Cell Transplantation Improves Outcome in Myelodysplastic Syndrome Across High-Risk Genetic Subgroups:Genetic Analysis of the Blood and Marrow Transplant Clinical Trials Network 1102 Study

PURPOSE:Allogeneic hematopoietic cell transplantation (HCT) in patients with myelodysplastic syndrome (MDS) improves overall survival (OS). We evaluated the impact of MDS genetics on the benefit of HCT in a biological assignment (donor v no donor) study.METHODS:We performed targeted sequencing in 309 patients age 50-75 years with International Prognostic Scoring System (IPSS) intermediate-2 or high-risk MDS, enrolled in the Blood and Marrow Transplant Clinical Trials Network 1102 study and assessed the association of gene mutations with OS. Patients with TP53 mutations were classified as TP53multihit if two alleles were altered (via point mutation, deletion, or copy-neutral loss of heterozygosity).RESULTS:The distribution of gene mutations was similar in the donor and no donor arms, with TP53 (28% v 29%; P =.89), ASXL1 (23% v 29%; P =.37), and SRSF2 (16% v 16%; P =.99) being most common. OS in patients with a TP53 mutation was worse compared with patients without TP53 mutation (21% ± 5% [SE] v 52% ± 4% at 3 years; P &lt;.001). Among those with a TP53 mutation, OS was similar between TP53single versus TP53multihit (22% ± 8% v 20% ± 6% at 3 years; P =.31). Considering HCT as a time-dependent covariate, patients with a TP53 mutation who underwent HCT had improved OS compared with non-HCT treatment (OS at 3 years: 23% ± 7% v 11% ± 7%; P =.04), associated with a hazard ratio of 3.89; 95% CI, 1.87 to 8.12; P &lt;.001 after adjustment for covariates. OS among patients with molecular IPSS (IPSS-M) very high risk without a TP53 mutation was significantly improved if they had a donor (68% ± 10% v 0% ± 12% at 3 years; P =.001).CONCLUSION:HCT improved OS compared with non-HCT treatment in patients with TP53 mutations irrespective of TP53 allelic status. Patients with IPSS-M very high risk without a TP53 mutation had favorable outcomes when a donor was available.</p

Características clínicas, microbiología y resultados de una cohorte de pacientes tratados con ceftolozane/tazobactam en centros de hospitalización de cuidados agudos, Houston, Texas, EE.UU

Antecedentes Ceftolozane/tazobactam es una combinación de β-lactámico/β-inhibidor de lactamasa con actividad contra una variedad de bacterias Gram-negativas, incluyendo Pseudomonas aeruginosa MDR. Este agente está aprobado para la neumonía bacteriana adquirida en el hospital y asociada a la ventilación mecánica. Sin embargo, la mayoría de los datos de resultados en el mundo real proceden de pequeñas cohortes observacionales. Por lo tanto, se trató de evaluar la utilización de ceftolozane/tazobactam en múltiples hospitales terciarios en Houston, TX, EE.UU.. Métodos Realizamos un estudio retrospectivo multicéntrico de pacientes que recibieron al menos 48 h de terapia con ceftolozano/tazobactam desde enero de 2016 hasta septiembre de 2019 en dos sistemas hospitalarios en Houston. Se recopilaron datos demográficos, clínicos y microbiológicos, incluido el aislado bacteriano infectante, cuando estaba disponible. El resultado primario fue el éxito clínico compuesto al alta hospitalaria. Los resultados secundarios incluyeron la mortalidad intrahospitalaria y la disposición clínica a los 14 y 30 días después del inicio de ceftolozane/tazobactam. Se utilizó un análisis de regresión logística multivariable para identificar los factores predictivos del resultado primario y la mortalidad. Los aislados recuperados se sometieron a pruebas de sensibilidad a ceftolozano/tazobactam y a WGS. Resultados Se incluyó a un total de 263 pacientes, y se alcanzó el éxito clínico compuesto en 185 pacientes (70,3%). La gravedad de la enfermedad fue el factor predictivo más consistente del éxito clínico. El tratamiento combinado con ceftolozane/tazobactam y otro agente Gram negativo activo se asoció a una reducción de las probabilidades de éxito clínico (OR 0,32; IC del 95%: 0,16-0,63). Se observó resistencia a ceftolozano/tazobactam en el 15,4% de los aislados disponibles para WGS; las mutaciones en ampC y ftsI fueron frecuentes pero no se agruparon con una ST concreta. Conclusiones La tasa de éxito clínico entre esta cohorte de pacientes tratados con ceftolozane/tazobactam fue similar en comparación con experiencias anteriores. Ceftolozane/tazobactam sigue siendo un agente alternativo para el tratamiento de aislados susceptibles de P. aeruginosaBackground Ceftolozane/tazobactam is a β-lactam/β-lactamase inhibitor combination with activity against a variety of Gram-negative bacteria, including MDR Pseudomonas aeruginosa. This agent is approved for hospital-acquired and ventilator-associated bacterial pneumonia. However, most real-world outcome data come from small observational cohorts. Thus, we sought to evaluate the utilization of ceftolozane/tazobactam at multiple tertiary hospitals in Houston, TX, USA. Methods We conducted a multicentre retrospective study of patients receiving at least 48 h of ceftolozane/tazobactam therapy from January 2016 through to September 2019 at two hospital systems in Houston. Demographic, clinical and microbiological data were collected, including the infecting bacterial isolate, when available. The primary outcome was composite clinical success at hospital discharge. Secondary outcomes included in-hospital mortality and clinical disposition at 14 and 30 days post ceftolozane/tazobactam initiation. Multivariable logistic regression analysis was used to identify predictors of the primary outcome and mortality. Recovered isolates were tested for susceptibility to ceftolozane/tazobactam and underwent WGS. Results A total of 263 patients were enrolled, and composite clinical success was achieved in 185 patients (70.3%). Severity of illness was the most consistent predictor of clinical success. Combination therapy with ceftolozane/tazobactam and another Gram-negative-active agent was associated with reduced odds of clinical success (OR 0.32, 95% CI 0.16–0.63). Resistance to ceftolozane/tazobactam was noted in 15.4% of isolates available for WGS; mutations in ampC and ftsI were common but did not cluster with a particular ST. Conclusions Clinical success rate among this patient cohort treated with ceftolozane/tazobactam was similar compared with previous experiences. Ceftolozane/tazobactam remains an alternative agent for treatment of susceptible isolates of P. aeruginosa

'To live and die [for] Dixie': Irish civilians and the Confederate States of America

Around 20,000 Irishmen served in the Confederate army in the Civil War. As a result, they left behind, in various Southern towns and cities, large numbers of friends, family, and community leaders. As with native-born Confederates, Irish civilian support was crucial to Irish participation in the Confederate military effort. Also, Irish civilians served in various supporting roles: in factories and hospitals, on railroads and diplomatic missions, and as boosters for the cause. They also, however, suffered in bombardments, sieges, and the blockade. Usually poorer than their native neighbours, they could not afford to become 'refugees' and move away from the centres of conflict. This essay, based on research from manuscript collections, contemporary newspapers, British Consular records, and Federal military records, will examine the role of Irish civilians in the Confederacy, and assess the role this activity had on their integration into Southern communities. It will also look at Irish civilians in the defeat of the Confederacy, particularly when they came under Union occupation. Initial research shows that Irish civilians were not as upset as other whites in the South about Union victory. They welcomed a return to normalcy, and often 'collaborated' with Union authorities. Also, Irish desertion rates in the Confederate army were particularly high, and I will attempt to gauge whether Irish civilians played a role in this. All of the research in this paper will thus be put in the context of the Drew Gilpin Faust/Gary Gallagher debate on the influence of the Confederate homefront on military performance. By studying the Irish civilian experience one can assess how strong the Confederate national experiment was. Was it a nation without a nationalism

Prediction of HLA genotypes from single-cell transcriptome data

The human leukocyte antigen (HLA) locus plays a central role in adaptive immune function and has significant clinical implications for tissue transplant compatibility and allelic disease associations. Studies using bulk-cell RNA sequencing have demonstrated that HLA transcription may be regulated in an allele-specific manner and single-cell RNA sequencing (scRNA-seq) has the potential to better characterize these expression patterns. However, quantification of allele-specific expression (ASE) for HLA loci requires sample-specific reference genotyping due to extensive polymorphism. While genotype prediction from bulk RNA sequencing is well described, the feasibility of predicting HLA genotypes directly from single-cell data is unknown. Here we evaluate and expand upon several computational HLA genotyping tools by comparing predictions from human single-cell data to gold-standard, molecular genotyping. The highest 2-field accuracy averaged across all loci was 76% by arcasHLA and increased to 86% using a composite model of multiple genotyping tools. We also developed a highly accurate model (AUC 0.93) for predicting HLA-DRB345 copy number in order to improve genotyping accuracy of the HLA-DRB locus. Genotyping accuracy improved with read depth and was reproducible at repeat sampling. Using a metanalytic approach, we also show that HLA genotypes from PHLAT and OptiType can generate ASE ratios that are highly correlated (R2 = 0.8 and 0.94, respectively) with those derived from gold-standard genotyping

Molecular Measurable Residual Disease Testing of Blood During AML Cytotoxic Therapy for Early Prediction of Clinical Response

Measurable residual disease (MRD) testing after initial chemotherapy treatment can predict relapse and survival in acute myeloid leukemia (AML). However, it has not been established if repeat molecular or genetic testing during chemotherapy can offer information regarding the chemotherapy sensitivity of the leukemic clone. Blood from 45 adult AML patients at day 1 and 4 of induction (n = 35) or salvage (n = 10) cytotoxic chemotherapy was collected for both quantitative real-time PCR (qPCR) assessment (WT1) and next generation sequencing (&gt;500 × depth) of 49 gene regions recurrently mutated in MDS/AML. The median age of subjects was 62 (23–78); 42% achieved a complete response. WT1 was overexpressed in most patients tested but was uninformative for very early MRD assessment. A median of 4 non-synonymous variants (range 0–7) were detected by DNA sequencing of blood on day 1 of therapy [median variant allele frequency (VAF): 29%]. Only two patients had no variants detectable. All mutations remained detectable in blood on day 4 of intensive chemotherapy and remarkably the ratio of mutated to wild-type sequence was often maintained. This phenomenon was not limited to variants in DNMT3A, TET2, and ASXL1. The kinetics of NPM1 and TP53 variant burden early during chemotherapy appeared to be exceptions and exhibited consistent trends in this cohort. In summary, molecular testing of blood on day 4 of chemotherapy is not predictive of clinical response to cytotoxic induction therapy in AML. The observed stability in variant allele frequency suggests that cytotoxic therapy may have a limited therapeutic index for clones circulating in blood containing these mutations. Further validation is required to confirm the utility of monitoring NPM1 and TP53 kinetics in blood during cytotoxic therapy

Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ.

Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63+TCF7+ myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63+TCF7+ myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression