The Use of Joint Models in Analysis of Aggregate Endpoints in RERF Cohort Studies.

In radiation risk estimation based on the Radiation Effects Research Foundation (RERF) cohort studies, one common analysis is Poisson regression on radiation dose and background and effect modifying variables of an aggregate endpoint such as all solid cancer incidence or all non-cancer mortality. As currently performed, these analyses require selection of a surrogate radiation organ dose, (e.g., colon dose), which could conceptually be problematic since the aggregate endpoint comprises events arising from a variety of organs. We use maximum likelihood theory to compare inference from the usual aggregate endpoint analysis to analyses based on joint analysis. These two approaches are also compared in a re-analysis of RERF Life Span Study all cancer mortality. We show that, except for a trivial difference, these two analytic approaches yield identical inference with respect to radiation dose response and background and effect modification when based on a single surrogate organ radiation dose. When repeating the analysis with organ-specific doses, an interesting issue of bias in intercept parameters arises when dose estimates are undefined for one sex when sex-specific outcomes are included in the aggregate endpoint, but a simple correction will avoid this issue. Lastly, while the joint analysis formulation allows use of organ-specific doses, the interpretation of such an analysis for inference regarding an aggregate endpoint can be problematic. To the extent that analysis of radiation risk for an aggregate endpoint is of interest, the joint-analysis formulation with a single surrogate dose is an appropriate analytic approach, whereas joint analysis with organ-specific doses may only be interpretable if endpoints are considered separately for estimating dose response. However, for neither approach is inference about dose response well defined.

Effect of radiation exposure on survival after first solid cancer diagnosis in A-bomb survivors.

BACKGROUND: Comparison of the estimated effect of atomic bomb radiation exposure on solid cancer incidence and solid cancer mortality in the RERF Life Span Study (LSS) reveals a difference in the magnitude and shape of the excess relative risk dose response. A possible contributing factor to this difference is pre-diagnosis radiation effect on post-diagnosis survival. Pre-diagnosis radiation exposure theoretically could influence post-diagnosis survival by affecting the genetic makeup and possibly aggressiveness of cancer, or by compromising tolerance for aggressive treatment for cancer. METHODS: We analyze the radiation effect on post-diagnosis survival in 20,463 LSS subjects diagnosed with first-primary solid cancer between 1958 and 2009 with particular attention to whether death was caused by the first-primary cancer, other cancer, or non-cancer diseases. RESULTS: From multivariable Cox regression analysis of cause-specific survival, the excess hazard at 1 Gy (EH1Gy) for death from the first primary cancer was not significantly different from zero - p = 0.23, EH1Gy = 0.038 (95 % CI: -0.023, 0.104). Death from other cancer and death from non-cancer diseases both were significantly associated with radiation dose: other cancer EH1Gy = 0.38 (95 % CI: 0.24, 0.53); non-cancer EH1Gy = 0.24 (95 % CI: 0.13, 0.36), both p < 0.001. CONCLUSION: There is no detectable large effect of pre-diagnosis radiation exposure on post-diagnosis death from the first primary cancer in A-bomb survivors. IMPACT: A direct effect of pre-diagnosis radiation exposure on cancer prognosis is ruled out as an explanation for the difference in incidence and mortality dose response in A-bomb survivors.

The Association of Radiation Exposure with Stable Chromosome Aberrations in Atomic Bomb Survivors Based on DS02R1 Dosimetry and FISH Methods.

The frequency of stable chromosome aberrations (sCA) in lymphocytes is a recognized radiation biological dosimeter. Its analysis can provide insights into factors that affect individual susceptibility as well as into the adequacy of radiation dose estimates used in studies of atomic bomb survivors. We analyzed the relationship between atomic bomb radiation exposure using the most recent DS02R1 dose estimates and the frequency of sCA as determined by FISH in 1,868 atomic bomb survivors. We investigated factors that may affect the background sCA rate and the shape and magnitude of the dose response. As in previous analyses of sCA in atomic bomb survivors that were based on Giemsa staining methods and used older DS86 dose estimates, the relationship between radiation dose and sCA rate was significant (P < 0.0001) with a linear-quadratic relationship at lower doses that did not persist at higher doses. As before, age at the time of the bombing and type of radiation shielding were significant dose-effect modifiers (P < 0.0001), but in contrast the difference in dose response by city was not so pronounced (P = 0.026) with a city effect not evident at doses below 1.25Gy. Background sCA rate increased with age at the time of examination (P < 0.0001), but neither sex, city, nor smoking was significantly associated with background rate. Based on FISH methods and recent dosimetry, the relationship between radiation dose and sCA frequency is largely consistent with previous findings, although the lesser importance of city as an effect modifier may reflect better dosimetry as well as more reproducible scoring of sCA. The persisting difference in sCA dose response by shielding category points to remaining problems with the accuracy or precision of radiation dose estimates in some A-bomb survivors.

Nelarabine, etoposide, and cyclophosphamide in relapsed pediatric T-acute lymphoblastic leukemia and T-lymphoblastic lymphoma (study T2008-002 NECTAR).

Children with relapse of T-cell acute lymphoblastic leukemia (T-ALL) or lymphoblastic lymphoma (T-LBL) have a dismal prognosis, largely due to difficulty attaining second remission. We hypothesized that adding etoposide and cyclophosphamide to the nucleoside analog nelarabine could improve response rates over single-agent nelarabine for relapsed T-ALL and T-LBL. This phase I dose-escalation trial's primary objective was to evaluate the dose and safety of nelarabine given in combination with etoposide at 100 mg/m2 /day and cyclophosphamide at 330-400 mg/m2 /day, each for 5 consecutive days in children with either T-ALL (13 patients) or T-LBL (10 patients). Twenty-three patients were treated at three dose levels; 21 were evaluable for dose-limiting toxicities (DLT) and response. The recommended phase II doses (RP2D) for this regimen, when given daily ×5 every 3 weeks, were nelarabine 650 mg/m2 /day, etoposide 100 mg/m2 /day, and cyclophosphamide 400 mg/m2 /day. DLTs included peripheral motor and sensory neuropathies. An expansion cohort to evaluate responses at the RP2D was terminated early due to slow accrual. The overall best response rate was 38% (8/21), with 33% (4/12) responses in the T-ALL cohort and 44% (4/9) responses in the T-LBL cohort. These response rates are comparable to those seen with single-agent nelarabine in this setting. These data suggest that the addition of cyclophosphamide and etoposide to nelarabine does not increase the incidence of neurologic toxicities or the response rate beyond that obtained with single-agent nelarabine in children with first relapse of T-ALL and T-LBL.

Expression of neuroblastoma-related genes in bone marrow at end of high-risk neuroblastoma therapy.

BACKGROUND: Minimal disease quantification may predict event-free survival (EFS) and overall survival (OS). METHODS: We evaluated mRNA expression of five neuroblastoma-associated genes (NB5 assay) in bone marrows (BM) of patients with newly diagnosed high-risk neuroblastoma who received consistent immunotherapy. mRNA expression of CHGA, DCX, DDC, PHOX2B, and TH genes in BM of 479 patients enrolled on the immunotherapy arm of Children's Oncology Group trials ANBL0032 and ANBL0931 was evaluated using real-time polymerase chain reaction (PCR)-based TaqMan low-density array. Results from end-consolidation and end-therapy were analyzed for association with five-year EFS/OS and patient and tumor characteristics. Tests of statistical significance were two-sided. RESULTS: NB5 assay detected neuroblastoma-related mRNA in 222 of 286 (77.6%) of BMs obtained at end-consolidation and 188 of 304 (61.8%) at end-therapy. Any mRNA level detected in end-therapy BM correlated with significantly worse EFS (57% [49.6%-63.7%] vs 73.0% [63.5%-80.4%]; P = 0.005), but not OS. Analysis limited to patients in complete response at end-therapy still found a significant difference in EFS with detectable versus not detectable NB5 assay results (58.9% [49.5%-67.1%] vs 76.6% [66.1%-84.2%]; P = 0.01). End-consolidation results did not correlate with EFS or OS. Multivariable analysis determined end-therapy NB5 assay BM results (P = 0.02), age at diagnosis (P = 0.002), and preconsolidation response (P = 0.02) were significantly associated with EFS independent of other clinical and biological parameters evaluated, including end-therapy response. CONCLUSIONS: If further validated in additional patient cohorts, the NB5 assay's ability to independently predict EFS from end-therapy could improve patient stratification for novel maintenance therapy trials after current end-therapy to improve outcome.

Temsirolimus combined with cyclophosphamide and etoposide for pediatric patients with relapsed/refractory acute lymphoblastic leukemia: a Therapeutic Advances in Childhood Leukemia Consortium trial (TACL 2014-001).

Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is commonly dysregulated in acute lymphoblastic leukemia (ALL). The TACL2014-001 phase I trial of the mTOR inhibitor temsirolimus in combination with cyclophosphamide and etoposide was performed in children and adolescents with relapsed/refractory ALL. Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily on days 1-5. The starting dose of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). PI3K/mTOR pathway inhibition was measured by phosphoflow cytometry analysis of peripheral blood specimens from treated patients. Sixteen heavily-pretreated patients were enrolled with 15 evaluable for toxicity. One dose-limiting toxicity of grade 4 pleural and pericardial effusions occurred in a patient treated at DL3. Additional dose-limiting toxicities were not seen in the DL3 expansion or DL4 cohort. Grade 3/4 non-hematologic toxicities occurring in three or more patients included febrile neutropenia, elevated alanine aminotransferase, hypokalemia, mucositis, and tumor lysis syndrome and occurred across all doses. Response and complete were observed at all dose levels with a 47% overall response rate and 27% complete response rate. Pharmacodynamic correlative studies demonstrated dose-dependent inhibition of PI3K/mTOR pathway phosphoproteins in all studied patients. Temsirolimus at doses up to 25 mg/m2 with cyclophosphamide and etoposide had an acceptable safety profile in children with relapsed/refractory ALL. Pharmacodynamic mTOR target inhibition was achieved and appeared to correlate with temsirolimus dose. Future testing of next-generation PI3K/mTOR pathway inhibitors with chemotherapy may be warranted to increase response rates in children with relapsed/refractory ALL.

Independent Assessment of the Children's Hepatic Tumors International Collaboration Risk Stratification for Hepatoblastoma and the Association of Tumor Histological Characteristics With Prognosis.

Importance: Hepatoblastoma is the most common pediatric liver malignant neoplasm, and accurate risk stratification is essential for guiding treatment. Objective: To validate the Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) in an independent cohort of patients with hepatoblastoma and evaluate the association of pretreatment hepatoblastoma histological subtype with prognosis. Design, Setting, and Participants: This is a single-institution retrospective cohort study of 96 pediatric patients with hepatoblastoma diagnosed and treated between June 1, 2000, and December 31, 2016, with recent therapy and independent of the CHIC-HS discovery cohort. Each patient was assigned a risk group according to CHIC-HS. The histological characteristics of each tumor were assessed based on the International Pediatric Liver Tumor Consensus Classification. Data were analyzed from May 2018 to May 2019. Main Outcomes And Measures: The main outcomes were event-free survival (EFS) and overall survival (OS). Cox regression analysis was used to examine the associations of patient characteristics and tumor histological characteristics with survival. Results: A total of 96 patients (median [range] age, 1.9 [0.4-18] years; 36 [38%] girls and 60 [63%] boys) were assessed, including 15 with very low risk, 28 with low risk, 23 with intermediate risk, and 30 with high risk, according to CHIC-HS criteria. There were a total of 13 cancer-related deaths; median (range) follow-up was 3.5 (0.1-17.8) years for those alive at the last follow-up. The estimated 5-year OS rates were 100% in the very low-risk group, 94.7% (95% CI, 68.1%-99.2%) in the low-risk group, 89.2% (95% CI, 63.1%-97.2%) in the intermediate-risk group, and 57.9% (95% CI, 34.6%-75.5%) in the high-risk group. In a multivariable analysis, we confirmed that CHIC-HS significantly estimated EFS (high-risk group vs very low- and low-risk groups: hazard ratio [HR], 45.59; 95% CI, 9.39-209.5; P < .001) and OS (high-risk group vs very low- and low-risk groups: HR, 21.95; 95% CI, 2.76-174.29; P < .001). In the subcohort of 84 patients for whom pretreatment tumor histological data were available, tumor epithelial histological subtypes were found to be significantly associated with both EFS and OS. Patients in the CHIC-HS high-risk group and with embryonal-only histological subtype had the highest risk of relapse or disease progression (high-risk: HR, 42.62; 95% CI, 9.91-203.9; embryonal: HR, 3.28; 95% CI, 1.21-8.9) and death (high-risk: HR, 18.78; 95% CI, 2.31-152.84; embryonal: HR, 7.12; 95% CI, 1.51-33.52). Conclusions and Relevance: This cohort study found that CHIC-HS performed as expected in an independent cohort that was more recently treated. Incorporation of pretreatment tumor histological data into CHIC-HS may provide additional prognostic value.

Decitabine and vorinostat with FLAG chemotherapy in pediatric relapsed/refractory AML: Report from the therapeutic advances in childhood leukemia and lymphoma (TACL) consortium.

Survival outcomes for relapsed/refractory pediatric acute myeloid leukemia (R/R AML) remain dismal. Epigenetic changes can result in gene expression alterations which are thought to contribute to both leukemogenesis and chemotherapy resistance. We report results from a phase I trial with a dose expansion cohort investigating decitabine and vorinostat in combination with fludarabine, cytarabine, and G-CSF (FLAG) in pediatric patients with R/R AML [NCT02412475]. Thirty-seven patients enrolled with a median age at enrollment of 8.4 (range, 1-20) years. There were no dose limiting toxicities among the enrolled patients, including two patients with Down syndrome. The recommended phase 2 dose of decitabine in combination with vorinostat and FLAG was 10 mg/m2 . The expanded cohort design allowed for an efficacy evaluation and the overall response rate among 35 evaluable patients was 54% (16 complete response (CR) and 3 complete response with incomplete hematologic recovery (CRi)). Ninety percent of responders achieved minimal residual disease (MRD) negativity (<0.1%) by centralized flow cytometry and 84% (n = 16) successfully proceeded to hematopoietic stem cell transplant. Two-year overall survival was 75.6% [95%CI: 47.3%, 90.1%] for MRD-negative patients vs. 17.9% [95%CI: 4.4%, 38.8%] for those with residual disease (p < .001). Twelve subjects (34%) had known epigenetic alterations with 8 (67%) achieving a CR, 7 (88%) of whom were MRD negative. Correlative pharmacodynamics demonstrated the biologic activity of decitabine and vorinostat and identified specific gene enrichment signatures in nonresponding patients. Overall, this therapy was well-tolerated, biologically active, and effective in pediatric patients with R/R AML, particularly those with epigenetic alterations.

Incidence of Diabetes in the Atomic Bomb Survivors: 1969-2015.

CONTEXT: Recent epidemiological studies have shown increased risk of diabetes among childhood cancer survivors who received high therapeutic doses of radiation, particularly to the total body or to the abdomen. However, the effect of low-to-moderate dose radiation (<4 Gy) on the risk of diabetes is still unknown. OBJECTIVES: To investigate the radiation effect on diabetes incidence among atomic bomb (A-bomb) survivors, and whether the dose response is modified by other factors including city, sex, and age at time of bombing (ATB). METHODS: 9131 participants without diabetes at baseline were observed through biennial clinical exams from 1969 to 2015. A Cox proportional hazards model was used to estimate hazard ratios (HR) to evaluate the dose response for diabetes incidence. RESULTS: During the study period, 1417 incident diabetes cases were identified. The overall crude incidence rate was 7.01/103 person-years. Radiation dose was significantly associated with diabetes incidence, with effect modification by city and age ATB. In Hiroshima, at ages 10 and 30 ATB, the HRs at 1 Gy of pancreatic radiation dose were 1.47 (95% CI, 1.31-1.66) and 1.13 (95% CI, 0.97-1.31), respectively. However, no significant radiation dose response was observed at these ages in Nagasaki. The HR for radiation dose was higher among those who were younger ATB and decreased 1% for each additional year of age. CONCLUSIONS: Among A-bomb survivors, a radiation association was suggested for incidence of diabetes. Results were inconsistent by city and age ATB, which could indicate potential confounding of the radiation association with diabetes.

Individual red blood cell nitric oxide production in sickle cell anemia: Nitric oxide production is increased and sickle shaped cells have unique morphologic change compared to discoid cells.

Sickle cell anemia (SCA) is characterized by decreased red blood cell (RBC) deformability due to polymerization of deoxygenated hemoglobin, leading to abnormal mechanical properties of RBC, increased cellular adhesion, and microcirculatory obstruction. Prior work has demonstrated that NO• influences RBC hydration and deformability and is produced at a basal rate that increases under shear stress in normal RBC. Nevertheless, the origin and physiological relevance of nitric oxide (NO•) production and scavenging in RBC remains unclear. We aimed to assess the basal and shear-mediated production of NO• in RBC from SCA patients and control (CTRL) subjects. RBCs loaded with a fluorescent NO• detector, DAF-FM (4-Amino-5-methylamino- 2',7'-difluorofluorescein diacetate), were imaged in microflow channels over 30-min without shear stress, followed by a 30-min period under 0.5Pa shear stress. We utilized non-specific nitric oxide synthase (NOS) blockade and carbon monoxide (CO) saturation of hemoglobin to assess the contribution of NOS and hemoglobin, respectively, to NO• production. Quantification of DAF-FM fluorescence intensity in individual RBC showed an increase in NO• in SCA RBC at the start of the basal period; however, both SCA and CTRL RBC increased NO• by a similar quantity under shear. A subpopulation of sickle-shaped RBC exhibited lower basal NO• production compared to discoid RBC from SCA group, and under shear became more circular in the direction of shear when compared to discoid RBC from SCA and CTRL, which elongated. Both CO and NOS inhibition caused a decrease in basal NO• production. Shear-mediated NO• production was decreased by CO in all RBC, but was decreased by NOS blockade only in SCA. In conclusion, total NO• production is increased and shear-mediated NO• production is preserved in SCA RBC in a NOS-dependent manner. Sickle shaped RBC with inclusions have higher NO• production and they become more circular rather than elongated with shear.

Development of a Risk Model for Pediatric Hospital-Acquired Thrombosis: A Report from the Children's Hospital-Acquired Thrombosis Consortium.

OBJECTIVE: To identify pertinent clinical variables discernible on the day of hospital admission that can be used to assess risk for hospital-acquired venous thromboembolism (HA-VTE) in children. STUDY DESIGN: The Children's Hospital-Acquired Thrombosis Registry is a multi-institutional registry for all hospitalized participants aged 0-21 years diagnosed with a HA-VTE and non-VTE controls. A risk assessment model (RAM) for the development of HA-VTE using demographic and clinical VTE risk factors present at hospital admission was derived using weighted logistic regression and the least absolute shrinkage and selection (Lasso) procedure. The models were internally validated using 5-fold cross-validation. Discrimination and calibration were assessed using area under the receiver operating characteristic curve and Hosmer-Lemeshow goodness of fit, respectively. RESULTS: Clinical data from 728 cases with HA-VTE and 839 non-VTE controls, admitted between January 2012 and December 2016, were abstracted. Statistically significant RAM elements included age <1 year and 10-22 years, cancer, congenital heart disease, other high-risk conditions (inflammatory/autoimmune disease, blood-related disorder, protein-losing state, total parental nutrition dependence, thrombophilia/personal history of VTE), recent hospitalization, immobility, platelet count >350 K/µL, central venous catheter, recent surgery, steroids, and mechanical ventilation. The area under the receiver operating characteristic curve was 0.78 (95% CI 0.76-0.80). CONCLUSIONS: Once externally validated, this RAM will identify those who are at low-risk as well as the greatest-risk groups of hospitalized children for investigation of prophylactic strategies in future clinical trials.

Tricuspid regurgitant jet velocity and myocardial tissue Doppler parameters predict mortality in a cohort of patients with sickle cell disease spanning from pediatric to adult age groups - revisiting this controversial concept after 16 years of additional evidence.

Sickle cell disease (SCD) is a monogenic hemoglobinopathy associated with significant morbidity and mortality. Cardiopulmonary, vascular and sudden death are the reasons for the majority of young adult mortality in SCD. To better understand the clinical importance of multi-level vascular dysfunction, in 2009 we assessed cardiac function including tricuspid regurgitant jet velocity (TRV), tissue velocity in systole(S') and diastole (E'), inflammatory, rheologic and hemolytic biomarkers as predictors of mortality in patients with SCD. With up to 9 years of follow up, we determined survival in 95 children, adolescents and adults with SCD. Thirty-eight patients (40%) were less than 21 years old at initial evaluation. Survival and Cox proportional-hazards analysis were performed. There was 19% mortality in our cohort, with median age at death of 35 years. In the pediatric subset, there was 11% mortality during the follow up period. The causes of death included cardiovascular and pulmonary complications in addition to other end-organ failure. On Cox proportional-hazards analysis, our model predicts that a 0.1 m/s increase in TRV increases risk of mortality 3%, 1 cm/s increase in S' results in a 91% increase, and 1 cm/s decrease in E' results in a 43% increase in mortality. While excluding cardiac parameters, higher plasma free hemoglobin was significantly associated with risk of mortality (p=.049). In conclusion, elevated TRV and altered markers of cardiac systolic and diastolic function predict mortality in a cohort of adolescents and young adult patients with SCD. These predictors should be considered when counseling cardiovascular risk and therapeutic optimization at transition to adult providers.

Surveillance for radiation-related late effects in childhood cancer survivors: The impact of using volumetric dosimetry.

BACKGROUND: Radiation-related screening guidelines for survivors of childhood cancer currently use irradiated regions (IR) to determine risk for late effects. However, contemporary radiotherapy techniques utilize volumetric dosimetry (VD) to determine organ-specific exposures, which could inform need for late effect surveillance. METHODS: This cross-sectional cohort study involved patients treated for cancer using computerized tomography-planned irradiation at Children's Hospital Los Angeles from 2000-2016. Organs at risk were identified using both VD and IR. Under each method, Children's Oncology Group Long-Term Follow-Up Guidelines were applied to determine radiation-related potential late effects and their correlative recommended screening practices. Patients served as their own controls. Mean number of potential late effects per patient and recommended screening practices per patient per decade of follow-up were compared using paired t-tests; comparisons were adjusted for diagnosis and gender using random effects, repeated measure linear regression. RESULTS: In this cohort (n = 132), median age at end of treatment was 10.6 years (range, 1.4-20.4). Brain tumor was the most common diagnosis (45%) and head/brain the most common irradiated region (61%). Under IR and VD, the mean number of potential late effects flagged was 24.4 and 21.7, respectively (-11.3%, p < 0.001); concordance between the two methods was 6.1%. Under VD, the difference in mean number of recommended screening practices per patient was -7.4% in aggregate but as large as -37.0% for diagnostic imaging and procedures (p < 0.001 for both). CONCLUSION: Use of VD rather than IR is feasible and enhances precision of guideline-based screening for radiation-related late effects in long-term childhood cancer survivors.

N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses.

Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a "liquid biopsy" of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. IMPLICATIONS: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.

mRNA and miRNA Profiles of Exosomes from Cultured Tumor Cells Reveal Biomarkers Specific for HPV16-Positive and HPV16-Negative Head and Neck Cancer.

Human papillomavirus (HPV)(+) and HPV(-) head and neck cancer (HNC) cells' interactions with the host immune system are poorly understood. Recently, we identified molecular and functional differences in exosomes produced by HPV(+) vs. HPV(-) cells, suggesting that genetic cargos of exosomes might identify novel biomarkers in HPV-related HNCs. Exosomes were isolated by size exclusion chromatography from supernatants of three HPV(+) and two HPV(-) HNC cell lines. Paired cell lysates and exosomes were analyzed for messenger RNA (mRNA) by qRT-PCR and microRNA (miR) contents by nanostring analysis. The mRNA profiles of HPV(+) vs. HPV(-) cells were distinct, with EGFR, TP53 and HSPA1A/B overexpressed in HPV(+) cells and IL6, FAS and DPP4 in HPV(-) cells. The mRNA profiles of HPV(+) or HPV(-) exosomes resembled the cargo of their parent cells. miR expression profiles in cell lysates identified 8 miRs expressed in HPV(-) cells vs. 14 miRs in HPV(+) cells. miR-205-5p was exclusively expressed in HPV(+) exosomes, and miR-1972 was only detected in HPV(-) exosomes. We showed that HPV(+) and HPV(-) exosomes recapitulated the mRNA expression profiles of their parent cells. Expression of miRs was dependent on the HPV status, and miR-205-5p in HPV(+) and miR-1972 in HPV(-) exosomes emerge as potential discriminating HPV-associated biomarkers.

Progressive vasoconstriction with sequential thermal stimulation indicates vascular dysautonomia in sickle cell disease.

Persons with sickle cell disease (SCD) exhibit subjective hypersensitivity to cold and heat perception in experimental settings, and triggers such as cold exposure are known to precipitate vaso-occlusive crises by still unclear mechanisms. Decreased microvascular blood flow (MBF) increases the likelihood of vaso-occlusion by increasing entrapment of sickled red blood cells in the microvasculature. Because those with SCD have dysautonomia, we anticipated that thermal exposure would induce autonomic hypersensitivity of their microvasculature with an increased propensity toward vasoconstriction. We exposed 17 patients with SCD and 16 control participants to a sequence of predetermined threshold temperatures for cold and heat detection and cold and heat pain via a thermode placed on the right hand. MBF was measured on the contralateral hand by photoplethysmography, and cardiac autonomic balance was assessed by determining heart rate variability. Thermal stimuli at both detection and pain thresholds caused a significant decrease in MBF in the contralateral hand within seconds of stimulus application, with patients with SCD showing significantly stronger vasoconstriction (P = .019). Furthermore, patients with SCD showed a greater progressive decrease in blood flow than did the controls, with poor recovery between episodes of thermal stimulation (P = .042). They had faster vasoconstriction than the controls (P = .033), especially with cold detection stimulus. Individuals with higher anxiety also experienced more rapid vasoconstriction (P = .007). Augmented vasoconstriction responses and progressive decreases in perfusion with repeated thermal stimulation in SCD are indicative of autonomic hypersensitivity in the microvasculature. These effects are likely to increase red cell entrapment in response to clinical triggers such as cold or stress, which have been associated with vaso-occlusive crises in SCD.

A phase I study of panobinostat in children with relapsed and refractory hematologic malignancies.

Background: Panobinostat demonstrates activity against pediatric cancers in vitro. A phase I trial in children with refractory hematologic malignancies was conducted. Study design: The trial evaluated two schedules of oral panobinostat using 3 + 3 dose escalations in 28-day cycles. For children with leukemia, panobinostat was given once daily three days a week each week at 24, 30 and 34 mg/m2/day. For children with lymphoma, panobinostat was given once daily three days a week every other week at 16, 20 and 24 mg/m2/day. Cerebrospinal fluid (CSF) from Day 29 of the first cycle, when available, was evaluated for PK. The study was registered on clinicaltrials.gov (NCT01321346) Results: Twenty-two subjects enrolled with leukemia. Five enrolled at dose level 1, 6 at dose level 2, and 11 at dose level 3. There was one dose limiting toxicity (DLT) in the leukemia arm at dose level 3 (Grade 4 hypertriglyceridemia), but no maximum tolerated dose (MTD) was identified. No subjects required removal from protocol therapy for QTc prolongation. PK studies were available in 11 subjects with similar exposure in children as in adults. Four Day 29 CSF specimens were found to have panobinostat levels below the lower limit of quantification. Five subjects with lymphoma were enrolled and received study drug, and 4 were evaluable for DLT. A DLT was reported (Grade 3 enteritis) on the lymphoma arm. Conclusions: Panobinostat was tolerated in heavily pretreated pediatric subjects. Gastrointestinal effects were observed on this study. There were no cardiac findings. There were no responses.

Excellent outcome of young children with nodular desmoplastic medulloblastoma treated on "Head Start" III: a multi-institutional, prospective clinical trial.

BACKGROUND: "Head Start" III, was a prospective clinical trial using intensive induction followed by myeloablative chemotherapy and autologous hematopoietic cell rescue (AuHCR) to either avoid or reduce the dose/volume of irradiation in young children with medulloblastoma. METHODS: Following surgery, patients received 5 cycles of induction followed by myeloablative chemotherapy using carboplatin, thiotepa, and etoposide with AuHCR. Irradiation was reserved for children >6 years old at diagnosis or with residual tumor post-induction. RESULTS: Between 2003 and 2009, 92 children <10 years old with medulloblastoma were enrolled. Five-year event-free survival (EFS) and overall survival (OS) rates (±SE) were 46 ±â€…5% and 62 ±â€…5% for all patients, 61 ±â€…8% and 77 ±â€…7% for localized medulloblastoma, and 35 ±â€…7% and 52 ±â€…7% for disseminated patients. Nodular/desmoplastic (ND) medulloblastoma patients had 5-year EFS and OS (±SE) rates of 89 ±â€…6% and 89 ±â€…6% compared with 26 ±â€…6% and 53 ±â€…7% for classic and 38 ±â€…13% and 46 ±â€…14% for large-cell/anaplastic (LCA) medulloblastoma, respectively. In multivariate Cox regression analysis, histology was the only significant independent predictor of EFS after adjusting for stage, extent of resection, regimen, age, and sex (P <0.0001). Five-year irradiation-free EFS was 78 ±â€…8% for ND and 21 ±â€…5% for classic/LCA medulloblastoma patients. Myelosuppression was the most common toxicity, with 2 toxic deaths. Twenty-four survivors completed neurocognitive evaluation at a mean of 4.9 years post-diagnosis. IQ and memory scores were within average range overall, whereas processing speed and adaptive functioning were low-average. CONCLUSION: We report excellent survival and preservation of mean IQ and memory for young children with ND medulloblastoma using high-dose chemotherapy, with most patients surviving without irradiation.

Significance of minimal residual disease in pediatric mixed phenotype acute leukemia: a multicenter cohort study.

The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multicenter cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (<0.01%) remission in most patients (70%). EOI MRD positivity was predictive of 5-year EFS (HR = 6.00, p < 0.001) and OS (HR = 9.57, p = 0.003). Patients who cleared MRD by EOC had worse survival compared with those EOI MRD negative. In contrast to adults with MPAL, ALL therapy without transplantation was adequate to treat most pediatric patients. Earlier MRD clearance was associated with better treatment success and survival. Prospective trials are now necessary to validate and refine MRD thresholds within the pediatric MPAL population and to identify salvage strategies for those with poor predicted survival.