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Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P

Alternative DNA structures that differ from the canonical B-DNA double helix, including Z-DNA, have received much attention recently due to their impact on DNA metabolic processes, including replication, transcription, and genome maintenance. Non-B-DNA-forming sequences can also stimulate genetic instability associated with disease development and evolution. Z-DNA can stimulate different types of genetic instability events in different species, and several different assays have been established to detect Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic and eukaryotic systems. In this chapter, we will introduce some of these methods including Z-DNA-induced mutation screening and detection of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Results from these assays should provide better insight into the mechanisms of Z-DNA-related genetic instability in different eukaryotic model systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Alternative DNA structure-forming sequences can stimulate mutagenesis and are enriched at mutation hotspots in human cancer genomes, implicating them in disease etiology. However, the mechanisms involved are not well characterized. Here, we discover that Z-DNA is mutagenic in yeast as well as human cells, and that the nucleotide excision repair complex, Rad10-Rad1(ERCC1-XPF), and the mismatch repair complex, Msh2-Msh3, are required for Z-DNA-induced genetic instability in yeast and human cells. Both ERCC1-XPF and MSH2-MSH3 bind to Z-DNA-forming sequences, though ERCC1-XPF recruitment to Z-DNA is dependent on MSH2-MSH3. Moreover, ERCC1-XPF - dependent DNA strand-breaks occur near the Z-DNA-forming region in human cell extracts, and we model these interactions at the sub-molecular level. We propose a relationship in which these complexes recognize and process Z-DNA in eukaryotes, representing a mechanism of Z-DNA-induced genomic instability.

Ewing Family Tumors (Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor) are common bone and soft tissue malignancies of childhood, adolescence and young adulthood. Chromosomal translocation in these tumors produces fusion oncogenes of the EWS/ETS class, with EWS/FLI1 being by far the most common. EWS/ETS chimera are the only well established driver mutations in these tumors and they function as aberrant transcription factors. Understanding the downstream genes whose expression is modified has been a central approach to the study of these tumors. FOXM1 is a proliferation associated transcription factor which has increasingly been found to play a role in the pathogenesis of a wide range of human cancers. Here we demonstrate that FOXM1 is expressed in Ewing primary tumors and cell lines. Reduction in FOXM1 expression in Ewing cell lines results in diminished potential for anchorage independent growth. FOXM1 expression is enhanced by EWS/FLI1, though, unlike other tumor systems, it is not driven by expression of the EWS/FLI1 target GLI1. Thiostrepton is a compound known to inhibit FOXM1 by direct binding. We show that Thiostrepton diminishes FOXM1 expression in Ewing cell lines and this reduction reduces cell viability through an apoptotic mechanism. FOXM1 is involved in Ewing tumor pathogenesis and may prove to be a useful therapeutic target in Ewing tumors.

Objective: Describe treatment patterns, resource use, and predictors of methylphenidate (MPH) switch among children (6-12 years), adolescents (13-17 years), and adults (≥ 18 years) with attention-deficit/hyperactivity disorder (ADHD)., Methods: This retrospective U.S. managed care database study used medical, pharmacy, and enrollment data to examine treatment patterns among patients with ≥ 1 ADHD diagnosis code (ICD-9 314.00-314.9), MPH pharmacy claims during 01/01/2004-09/30/2006, and no ADHD pharmacy claims in prior 6 months. Patients were followed for 1 year for dosage change, switch (change to non-MPH treatment), augmentation, persistence (number days on index medication) and adherence (days supplied/days persistent). End points were assessed by age group and MPH formulation. Cox proportional hazards modeling was conducted to determine predictors of MPH switch., Results: Among 23,860 MPH users, 51.4% had a dosing change, 14% switched to a non-MPH agent, and 4% augmented MPH therapy. Among those prescribed long-acting (LA) MPH (N = 14,681), switching rates were 14% for children, 13% for adolescents, and 16% for adults. Augmentation rates for LA MPH were <5%. Overall, 53% of patients were adherent with mean persistence of 219 days. For the subgroup of patients prescribed LA MPH (n = 14,681), adherence ranged from 49% (adolescents) to 59% (children); persistence varied between 183 days (adults) to 256 days (children). During the 1-year follow-up, office/clinic visits were the major driver of health care resource use in MPH patients (mean 9.7 visits/patient). Patients with psychiatric comorbidity utilized significantly greater services. Predictors of MPH switch included psychiatric comorbidity (hazards ratio [HR] 1.37; 95% confidence interval [CI] = 1.26-1.48; p < 0.0001) and specialty prescribers (HR 1.19, 95% CI = 1.04-1.35; p = 0.011). Potential limitations of this study include use of claims data for definition of drug usage; inclusion of medications approved for use in ADHD; assessment of switching that may not have captured short-term augmentation; absence of economic, clinical and other variables from the claims dataset that may have influenced treatment selection, and outcomes. The 6-month baseline period to determine newly treated patients may not guarantee exclusion of all previously treated patients who restart therapy after an extended period., Conclusions: Children exhibited the highest persistence of MPH users. ADHD patients on MPH therapy with a psychiatric comorbidity may require additional follow-up to help improve adherence and reduce health care resource use.

Purpose: To estimate patient- and episode-level direct costs of chronic obstructive pulmonary disease (COPD) among commercially insured patients in the US., Methods: In this retrospective claims-based analysis, commercial enrollees with evidence of COPD were grouped into five mutually exclusive cohorts based on the most intensive level of COPD-related care they received in 2006, ie, outpatient, urgent outpatient (outpatient care in addition to a claim for an oral corticosteroid or antibiotic within seven days), emergency department (ED), standard inpatient admission, and intensive care unit (ICU) cohorts. Patient- level COPD-related annual health care costs, including patient- and payer-paid costs, were compared among the cohorts. Adjusted episode-level costs were calculated., Results: Of the 37,089 COPD patients included in the study, 53% were in the outpatient cohort, 37% were in the urgent outpatient cohort, 3% were in the ED cohort, and the standard admission and ICU cohorts together comprised 6%. Mean (standard deviation, SD) annual COPD-related health care costs (2008 US$) increased across the cohorts (P < 0.001), ranging from $2003 ($3238) to $43,461 ($76,159) per patient. Medical costs comprised 96% of health care costs for the ICU cohort. Adjusted mean (SD) episode-level costs were $305 ($310) for an outpatient visit, $274 ($336) for an urgent outpatient visit, $327 ($65) for an ED visit, $9745 ($2968) for a standard admission, and $33,440 for an ICU stay., Conclusion: Direct costs of COPD-related care for commercially insured patients are driven by hospital stays with or without ICU care. Exacerbation prevention resulting in reduced need for inpatient care could lower costs.

Objective: To develop a descriptive profile of attention-deficit/hyperactivity disorder (ADHD) pharmacological treatment patterns in terms of persistence, adherence, augmentation, switching, and dosing changes; and to assess differences in treatment patterns with regard to ADHD medication type, class, and duration of action., Methods: This retrospective claims database analysis used medical data, pharmacy data, and enrollment information to examine treatment patterns among patients with at least one claim with a diagnosis code for ADHD and a filled prescription for ADHD medication (index therapy) during the period 01 January 2004 through 30 September 2006. Treatment persistence and adherence (days supplied/days persistent) were calculated. Dose changes, medication switching, and augmentation were analyzed at three levels of comparison: class (stimulant vs nonstimulant [atomoxetine]), drug type (amphetamine vs methylphenidate), and duration of action (short, intermediate, long). Statistical comparisons were made using the chi-square test for proportions and Student's t-test or the F-test from one-way ANOVA for means., Results: Of 60,010 patients meeting eligibility criteria, 58.4% were younger than age 18. Most (78.4%) were prescribed a stimulant as their index therapy. Persistence and adherence were greater for patients on stimulants (vs the nonstimulant), for patients on amphetamines (vs methylphenidates), and for patients on long-acting medications (vs short- and intermediate-acting medications; all p < 0.0001). Index drug dose changes were least likely among individuals taking the nonstimulant (vs stimulants), methylphenidates (vs amphetamines), or intermediate-acting drugs (vs short- and long-acting drugs; all p < 0.0001), and medication switches were more frequent among those on nonstimulants, methylphenidates, or short-acting drugs (all p < 0.0001). Subjects taking long-acting medication were less likely to augment with a drug with a different duration of action than those taking intermediate- or short-acting medication (p < 0.0001). This claims-based study is limited by possible discrepancies between claims and patient behaviors (i.e., a claim for a prescription does not necessarily indicate that the medication was taken as prescribed)., Conclusions: Patients were more stable on treatment compared with their respective comparator groups if their index therapy was a stimulant, long-acting drug, or amphetamine.

The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.

Background: Transplantation of nonrenal organs is often complicated by chronic renal disease with multifactorial causes. We conducted a population-based cohort analysis to evaluate the incidence of chronic renal failure, risk factors for it, and the associated hazard of death in recipients of nonrenal transplants., Methods: Pretransplantation and post-transplantation clinical variables and data from a registry of patients with end-stage renal disease (ESRD) were linked in order to estimate the cumulative incidence of chronic renal failure (defined as a glomerular filtration rate of 29 ml per minute per 1.73 m2 of body-surface area or less or the development of ESRD) and the associated risk of death among 69,321 persons who received nonrenal transplants in the United States between 1990 and 2000., Results: During a median follow-up of 36 months, chronic renal failure developed in 11,426 patients (16.5 percent). Of these patients, 3297 (28.9 percent) required maintenance dialysis or renal transplantation. The five-year risk of chronic renal failure varied according to the type of organ transplanted - from 6.9 percent among recipients of heart-lung transplants to 21.3 percent among recipients of intestine transplants. Multivariate analysis indicated that an increased risk of chronic renal failure was associated with increasing age (relative risk per 10-year increment, 1.36; P<0.001), female sex (relative risk among male patients as compared with female patients, 0.74; P<0.001), pretransplantation hepatitis C infection (relative risk, 1.15; P<0.001), hypertension (relative risk, 1.18; P<0.001), diabetes mellitus (relative risk, 1.42; P<0.001), and postoperative acute renal failure (relative risk, 2.13; P<0.001). The occurrence of chronic renal failure significantly increased the risk of death (relative risk, 4.55; P<0.001). Treatment of ESRD with kidney transplantation was associated with a five-year risk of death that was significantly lower than that associated with dialysis (relative risk, 0.56; P=0.02)., Conclusions: The five-year risk of chronic renal failure after transplantation of a nonrenal organ ranges from 7 to 21 percent, depending on the type of organ transplanted. The occurrence of chronic renal failure among patients with a nonrenal transplant is associated with an increase by a factor of more than four in the risk of death., (Copyright 2003 Massachusetts Medical Society)

Background: Graft-versus-host disease (GVHD) is an important, underdiagnosed cause of mortality associated with liver transplantation. We identified 12 cases of GVHD among 1,082 liver transplantations performed in patients at our institution between 1991 and 1998. Patients typically developed fever, skin rash, diarrhea, or pancytopenia within 2 to 6 weeks after their transplant. Treatment generally involved increased immune suppression and hematopoietic cytokines (granulocyte colony stimulating factor, granulocyte monocyte colony stimulating factor); however, all but one patient died, most often from sepsis. Early in its course, GVHD was difficult to distinguish from cytomegalovirus disease or drug reactions. The diagnosis was confirmed by demonstration of substantial donor lymphoid chimerism., Methods: To identify risk factors for severe GVHD, a retrospective analysis was performed comparing index cases with the rest of the cases in our institutional experience., Results: Closely matched human leukocyte antigen recipients, those older than 65 years, and recipients with donors more than 40 years younger were at higher risk for GVHD. One case occurred in a patient with a congenital immunodeficiency., Conclusions: Liver transplant-associated GVHD is a progressive and fatal disease. Future approaches should focus on prevention and might include avoidance of closely matched human leukocyte antigen donors, treatment of the donor to reduce the number of lymphocytes, or reduction of immunosuppression in the early posttransplant period.

Despite declining in-hospital deaths, deceased donor organ donation increased in all demographic segments of the US population from 1990 to 1999. Although the rate of growth in older donors was more dramatic, there was substantial growth in the most physiologically suitable donor age groups, which contribute most to the donor pool. Nationwide, the average annual growth in donation rate for in-hospital deaths ages 1-70 was 5.0%, with wide regional variation. While the national trend is patently inadequate to meet the need for organ transplantation, the wide range of programs aimed at improving organ donation at all levels of society point to an encouraging pattern that may have resulted, in part, from these initiatives.