Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas

Antifungal Prophylaxis and Risk for Invasive Mold Infections in Children with Hematologic Malignancies

Introduction: Invasive mold infections (IMI) are a leading cause of mortality in immunocompromised hosts. Children diagnosed with hematologic malignancies experience profound, prolonged neutropenia following intensive chemotherapy, and are at increased risk for infection-related outcomes. Depending on the anticipated therapeutic intensity, antimicrobial prophylaxis may be employed to mitigate risk for infection. We conducted a retrospective review of children diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or lymphoma between 2006-2015 and determined the incidence of IMI to be 4.8% (47/976), with an exceptionally high incidence observed in patients with AML (8.1%). This observation prompted a change in clinical practice that broadened prophylaxis for high risk patients to include coverage of molds, and resulted in development of a risk-stratified algorithm for antifungal prophylaxis in children with hematologic malignancies. The objective of this study was to evaluate the change in IMI incidence post-implementation of this algorithm, and to identify host factors contributing to risk for IMI in children with hematologic malignancies. Objective: The objective was to compare the incidence of IMI pre/post implementation of antifungal prophylaxis decision tree. Also, it was planned to evaluate the impact of race/ethnicity on the development of IMI in children with hematologic malignancies. Methods: We conducted a retrospective review of children ≤ 21 years old and diagnosed with ALL, AML, or lymphoma between 2016-2019, and were treated for IMI between 2016 and June 2020. To identify potential cases, we employed a strategy identical to the one used in the 2006-2015 review, specifically, a search of the electronic medical record utilizing ICD9 codes broadly inclusive of relevant cancer and fungal diagnoses. Each potentially eligible case was then reviewed for the following inclusion/exclusion criteria (also identical to the prior review): diagnosis and treatment of ALL, AML, or lymphoma at Texas Children’s Hospital, diagnosis of IMI that met criteria for ‘proven’ or ‘probable’ per the European Organization for Research and Treatment of Cancer/Mycoses Study Group and occurring prior to stem cell transplant, and no underlying immunodeficiency or history of solid organ transplant. Host and disease-related factors, as well as IMI incidence, were compared for 2006-2015 vs. 2016-2020 using a Chi-square, Fisher, or Student t-test as appropriate, and host factors predictive of IMI were assessed by multivariable linear regression. Results: The overall incidence of proven/probable IMI in children diagnosed with hematological malignancies between 2006-2019 was 4.2% (61/1456). The incidence of IMI decreased from 4.8% to 2.9% between 2006-2015 and 2016-2020. For specific diagnoses, the rate of IMI decreased from 5.0% to 3.6% (ALL, 35/705 vs. 10/276), from 1.9% to 1.4% (lymphoma, 47/976 vs. 14/480), and from 8.1% to 3.2% (AML, 9/111 vs. 2/62). No significant differences in host factor or disease-related characteristics were noted when comparing IMI cases in 2006-2015 vs. 2016-2020, nor were there differences in the proportion of patients in relapse at the time of IMI or taking antifungal prophylaxis. Substantial differences in representative mold species were noted between the two-time periods, e.g. Aspergillus spp. accounted for 19/47 IMI from 2006-2015, but accounted for none of the IMIs diagnosed 2016-2020. In 2016-2020, 5/14 IMI were due to Trichosporon spp., with 4/14 Rhizopus spp., 2/14 Fusarium spp., 1/14 Curvularia spp., 1/14 Histoplasma spp., and 1 that met criteria for probable IMI. In multivariable analyses (Table 1), Hispanics were more likely to develop an IMI than non-Hispanics (p=0.04, OR 1.94, CI 1.03-3.66), and those with lymphoma were less likely to develop an IMI than those with ALL (p=0.03, OR 0.33, CI 0.12-0.87). Patients diagnosed between 2016- 2019 were substantially less likely to develop IMI than those diagnosed 2006-2015 (p=0.003, OR 0.33, CI 0.16-0.69). Discussion and Conclusion: In this single institution study, risk for IMI in children with hematologic malignancies declined significantly after implementation of an antifungal prophylaxis algorithm that broadened coverage for high risk populations. Hispanics were at higher risk for IMI than non-Hispanics, suggesting a need to investigate relevant factors contributing to this disparity. This project can be used to further investigate the factors that contributed to invasive mold infections using a larger study populations. We can then continue to explore the potential contributing factors to the racial and ethnic disparities by including potential contributing factors such as socioeconomic factors and genetic risk

The performance of human papillomavirus high-risk DNA testing in the screening and diagnostic settings.

OBJECTIVE: We sought to evaluate the performance of the human papillomavirus high-risk DNA test in patients 30 years and older. MATERIALS AND METHODS: Screening (n=835) and diagnosis (n=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II (HCII) test result with the worst histologic report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR-), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear. RESULTS: For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR- were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR- were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing. CONCLUSIONS: The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older

Influence of Inflammatory and Oxidative Stress Pathways on Longitudinal Symptom Experiences in Children With Leukemia

Purpose: The purpose of this study was to explore the influence of oxidative stress (F2-isoprostanes) and inflammatory (interleukin [IL]-8) biomarkers on symptom trajectories during the first 18 months of childhood leukemia treatment. Method: A repeated-measures design was used to evaluate symptoms experienced by 218 children during treatment. A symptom cluster (fatigue, pain, and nausea) was explored over four time periods: initiation of post-induction therapy, 4 and 8 months into post-induction therapy, and the beginning of maintenance therapy (12 months postinduction). F2-isoprostanes and IL-8 were evaluated in cerebrospinal fluid (CSF) samples collected at baseline (diagnosis) and then at the four time periods. The longitudinal relationships of these biomarkers with the symptom cluster were examined using the longitudinal parallel process. Results: Pain and fatigue levels were highest during the post-induction phases of treatment and decreased slightly during maintenance therapy, while nausea scores were relatively stable. Even in the later phases of treatment, children continued to experience symptoms. CSF levels of the biomarkers increased during the post-induction phases of treatment. Early increases in the biomarkers were associated with more severe symptoms during the same period; patients who had increased biomarkers over time also experienced more severe symptoms over time. Conclusions: Findings reveal that children experienced symptoms throughout the course of leukemia treatment and support hypothesized longitudinal relationships of oxidative stress and inflammatory biomarkers with symptom severity. Activation of the biomarker pathways during treatment may explain underlying mechanisms of symptom experiences and identify which children are at risk for severe symptoms.National Institutes of Health [R01CA1693398]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Clinical Factors Associated with Long-Term Complete Remission versus Poor Response to Chemotherapy in HIV-Infected Children and Adolescents with Kaposi Sarcoma Receiving Bleomycin and Vincristine: A Retrospective Observational Study

Kaposi sarcoma (KS) is the most common HIV-associated malignancy in children and adolescents in Africa. Pediatric KS is distinct from adult disease. We evaluated the clinical characteristics associated with long-term outcomes. We performed a retrospective observational analysis of 70 HIV-infected children and adolescents with KS less than 18 years of age diagnosed between 8/2010 and 6/2013 in Lilongwe, Malawi. Local first-line treatment included bleomycin and vincristine plus nevirapine-based highly active anti-retroviral therapy (HAART). Median age was 8.6 years (range 1.7-17.9); there were 35 females (50%). Most common sites of presentation were: lymph node (74%), skin (59%), subcutaneous nodules (33%), oral (27%), woody edema (24%), and visceral (16%). Eighteen (26%) presented with lymphadenopathy only. Severe CD4 suppression occurred in 28%. At time of KS diagnosis, 49% were already on HAART. Overall, 28% presented with a platelet count \u3c 100 x 109/L and 37% with hemoglobin \u3c 8 g/dL. The 2-year event-free (EFS) and overall survival (OS) were 46% and 58% respectively (median follow-up 29 months, range 15-50). Multivariable analysis of risk of death and failure to achieve EFS demonstrated that visceral disease (odds ratios [OR] 19.08 and 11.61, 95% CI 2.22-163.90 and 1.60-83.95 respectively) and presenting with more than 20 skin/oral lesions (OR 9.57 and 22.90, 95% CI 1.01-90.99 and 1.00-524.13 respectively) were independent risk factors for both. Woody edema was associated with failure to achieve EFS (OR 7.80, 95% CI 1.84-33.08) but not death. Univariable analysis revealed that lymph node involvement was favorable for EFS (OR 0.28, 95% CI 0.08-0.99), while T1 TIS staging criteria, presence of cytopenias, and severe immune suppression were not associated with increased mortality. Long-term complete remission is achievable in pediatric KS, however outcomes vary according to clinical presentation. Based on clinical heterogeneity, treatment according to risk-stratification is necessary to improve overall outcomes

Hospital Costs Related to Early Extubation after Infant Cardiac Surgery

Background The Pediatric Heart Network Collaborative Learning Study (PHN CLS) increased early extubation rates after infant Tetralogy of Fallot (TOF) and coarctation (CoA) repair across participating sites by implementing a clinical practice guideline (CPG). The impact of the CPG on hospital costs has not been studied. Methods PHN CLS clinical data were linked to cost data from Children’s Hospital Association by matching on indirect identifiers. Hospital costs were evaluated across active and control sites in the pre- and post-CPG periods using generalized linear mixed effects models. A difference-in-difference approach was used to assess whether changes in cost observed in active sites were beyond secular trends in control sites. Results Data were successfully linked on 410/428 (96%) of eligible patients from 4 active and 4 control sites. Mean adjusted cost/case for TOF repair was significantly reduced in the post-CPG period at active sites ($42,833 vs.$56,304, p<0.01) and unchanged at control sites ($47,007 vs.$46,476, p=0.91), with an overall cost reduction of 27% in active vs. control sites (p=0.03). Specific categories of cost reduced in the TOF cohort included clinical (-66%, p<0.01), pharmacy (-46%, p=0.04), lab (-44%, p<0.01), and imaging (-32%, p<0.01). There was no change in costs for CoA repair at active or control sites. Conclusions The early extubation CPG was associated with a reduction in hospital costs for infants undergoing repair of TOF, but not CoA repair. This CPG represents an opportunity to both optimize clinical outcome and reduce costs for certain infant cardiac surgeries

Rare deleterious germline variants and risk of lung cancer

Recent studies suggest that rare variants exhibit stronger effect sizes and might play a crucial role in the etiology of lung cancers (LC). Whole exome plus targeted sequencing of germline DNA was performed on 1045 LC cases and 885 controls in the discovery set. To unveil the inherited causal variants, we focused on rare and predicted deleterious variants and small indels enriched in cases or controls. Promising candidates were further validated in a series of 26,803 LCs and 555,107 controls. During discovery, we identified 25 rare deleterious variants associated with LC susceptibility, including 13 reported in ClinVar. Of the five validated candidates, we discovered two pathogenic variants in known LC susceptibility loci, ATM p.V2716A (Odds Ratio [OR] 19.55, 95%CI 5.04–75.6) and MPZL2 p.I24M frameshift deletion (OR 3.88, 95%CI 1.71–8.8); and three in novel LC susceptibility genes, POMC c.*28delT at 3′ UTR (OR 4.33, 95%CI 2.03–9.24), STAU2 p.N364M frameshift deletion (OR 4.48, 95%CI 1.73–11.55), and MLNR p.Q334V frameshift deletion (OR 2.69, 95%CI 1.33–5.43). The potential cancer-promoting role of selected candidate genes and variants was further supported by endogenous DNA damage assays. Our analyses led to the identification of new rare deleterious variants with LC susceptibility. However, in-depth mechanistic studies are still needed to evaluate the pathogenic effects of these specific alleles

Focused Analysis of Exome Sequencing Data for Rare Germline Mutations in Familial and Sporadic Lung Cancer

AbstractIntroductionThe association between smoking-induced chronic obstructive pulmonary disease (COPD) and lung cancer (LC) is well documented. Recent genome-wide association studies (GWAS) have identified 28 susceptibility loci for LC, 10 for COPD, 32 for smoking behavior, and 63 for pulmonary function, totaling 107 nonoverlapping loci. Given that common variants have been found to be associated with LC in genome-wide association studies, exome sequencing of these high-priority regions has great potential to identify novel rare causal variants.MethodsTo search for disease-causing rare germline mutations, we used a variation of the extreme phenotype approach to select 48 patients with sporadic LC who reported histories of heavy smoking—37 of whom also exhibited carefully documented severe COPD (in whom smoking is considered the overwhelming determinant)—and 54 unique familial LC cases from families with at least three first-degree relatives with LC (who are likely enriched for genomic effects).ResultsBy focusing on exome profiles of the 107 target loci, we identified two key rare mutations. A heterozygous p.Arg696Cys variant in the coiled-coil domain containing 147 (CCDC147) gene at 10q25.1 was identified in one sporadic and two familial cases. The minor allele frequency (MAF) of this variant in the 1000 Genomes database is 0.0026. The p.Val26Met variant in the dopamine β-hydroxylase (DBH) gene at 9q34.2 was identified in two sporadic cases; the minor allele frequency of this mutation is 0.0034 according to the 1000 Genomes database. We also observed three suggestive rare mutations on 15q25.1: iron-responsive element binding protein neuronal 2 (IREB2); cholinergic receptor, nicotinic, alpha 5 (neuronal) (CHRNA5); and cholinergic receptor, nicotinic, beta 4 (CHRNB4).ConclusionsOur results demonstrated highly disruptive risk-conferring CCDC147 and DBH mutations