Hypertension Severity and Declines in Left Ventricular Ejection Fraction Among Women Receiving Adjuvant Chemotherapy for Breast Cancer (WF-97415 UPBEAT).

BACKGROUND: Hypertension is a risk factor for experiencing left ventricular ejection fraction (LVEF) declines during receipt of potentially cardiotoxic breast cancer (BC) treatment. We sought to determine whether the hypertension stage is associated with LVEF decline during BC treatment. METHODS: Across 24 centers, cardiac magnetic resonance measures of LVEF and brachial arterial blood pressure (BP) measurements were performed in women with stages I to III BC before and 3 months after initiating potentially cardiotoxic chemotherapy. Using multivariable analysis, we assessed in a blinded fashion the association between 3-month ΔLVEF and precancer treatment American Heart Association/American College of Cardiology stages of hypertension. RESULTS: Among 204 women, age averaged 56±1 years with 75% being White and 20% of Black race. Participants received anthracycline (45.6%), trastuzumab (22.5%), cyclophosphamide (52.9%), or paclitaxel (50%). After accounting for pretreatment LVEF, diabetes status, tobacco use, age, the number of antihypertensive medications, and body mass index, those with stage II hypertension experienced an LVEF decline of -2.89% ([95% CI, -0.69% to -5.19%]; P=0.01) relative to individuals with normal BP. Other stages saw nonsignificant declines relative to normal BP to elevated BP (-1.63% [95% CI, -0.62% to 3.88%]; P=0.16) and stage I hypertension (-0.94% [95% CI, -0.90% to 2.78%]; P=0.32). CONCLUSIONS: Compared with women receiving treatment for BC with normal BP, there is a stronger association of decline in LVEF in women with stage II hypertension relative to women with other hypertension stages. This raises the possibility that stage along with hypertension presence may be associated with an increased risk for the LVEF decline among women receiving potentially cardiotoxic chemotherapy for BC. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02791581 and NCT01719562.

Physical Activity During Breast Cancer Therapy Associates With Preserved Exercise Capacity and Cardiac Function (WF97415).

Background: Cancer treatment increases cardiovascular disease risk, but physical activity (PA) may prevent cardiovascular disease. Objectives: This study examined whether greater PA was associated with better submaximal exercise capacity and cardiac function during cancer therapy. Methods: Participants included 223 women with stage I to III breast cancer (BC) before and 3 months after undergoing treatment and 126 control participants. Leisure-time PA (LTPA) was reported using the Godin-Shephard LTPA questionnaire. Cardiac function was assessed by cardiac magnetic resonance. Submaximal exercise capacity was determined by 6-minute walk distance. Results: BC participants reported similar baseline LTPA scores (24.7; 95% CI: 21.7-28.0) as control participants (29.4; 95% CI: 25.0-34.2). The BC group declined to 16.9 (95% CI: 14.4-19.6) at 3 months relative to 30.8 (95% CI: 26.2-35.8) in control participants. Among BC participants, more LTPA was related to better exercise capacity (ß ± SE: 7.1 ± 1.6; 95% CI: 4.0-10.1) and left ventricular (LV) circumferential strain (-0.16 ± 0.07; 95% CI: -0.29 to -0.02). Increased LTPA over the 3 months was associated with decreased likelihood of treatment-induced cardiac dysfunction according to LV circumferential strain classifications (OR: 0.98; 95% CI: 0.97-0.998). BC participants reporting insufficient LTPA according to PA guidelines exhibited deteriorations in exercise capacity (adjusted mean difference ± SE: -29 ± 10 m; P = 0.029), LV end-systolic volume (5.8 ± 1.3 mL; P < 0.001), LV ejection fraction (-3.2% ± 0.8%; P = 0.002), and LV circumferential strain (2.5% ± 0.5%; P < 0.001), but BC participants meeting LTPA guidelines did not exhibit these adverse changes. Conclusions: PA declined during BC therapy; however, PA participation was associated with attenuated declines in exercise capacity and cardiac function that are often observed in this population. (Understanding and Predicting Breast Cancer Events After Treatment [WF97415 UPBEAT]; NCT02791581).

Statins and Left Ventricular Ejection Fraction Following Doxorubicin Treatment.

BACKGROUND: Statins taken for cardiovascular indications by patients with breast cancer and lymphoma during doxorubicin treatment may attenuate left ventricular ejection fraction (LVEF) decline, but the effect of statins on LVEF among patients with no cardiovascular indications is unknown. METHODS: A double-blind, placebo-controlled, 24-month randomized trial of 40 mg of atorvastatin per day administered to patients with breast cancer and lymphoma receiving doxorubicin was conducted within the National Cancer Institute Community Oncology Research Program across 31 sites in the United States. At pretreatment and then 6 and 24 months after initiating doxorubicin, we assessed left ventricular (LV) volumes, strain, mass, and LVEF through cardiac magnetic resonance imaging, along with cognitive function and serum markers of inflammation. The primary outcome was the difference in 24-month LVEF between placebo and treatment groups, adjusted for pretreatment LVEF. RESULTS: A total of 279 participants were enrolled in the trial. Participants had a mean (±SD) age of 49±12 years; 92% were women; and 83% were White. The mean (±SD) LVEF values were 61.7±5.5% before treatment and 57.4±6.8% at 24 months in the placebo group and 62.6±6.4% before treatment and 57.7±5.6% at 24 months in the atorvastatin group. On the basis of a multiple imputed data set for missing data and adjusted for each individual's pretreatment LVEF, 24-month declines in LVEF averaged 3.3±0.6 percentage points and 3.2±0.7 percentage points, for those randomly assigned to placebo versus statins, respectively (P=0.93). Across both treatment arms, similar percentages of individuals experienced changes of more than 10 percentage points in LVEF, LV strain, LV mass, cognition, and inflammation biomarkers, including among those with greater than 90% drug compliance. CONCLUSIONS: In patients with breast cancer and lymphoma with no existing indication for statin therapy, prospective statin administration did not affect LVEF declines 2 years after doxorubicin. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01988571.).

Both intermuscular fat and LVEF decline promote heart failure symptoms in cancer survivors.

BACKGROUND: Approximately 20% of cancer survivors treated with chemotherapy experience worsening heart failure (HF) symptoms post-cancer treatment. While research has predominantly investigated the role of cardiotoxic treatments, much less attention has focused on other risk factors, such as adiposity. However, emerging data in cancer survivors indicates that adiposity may also impact a variety of cardiovascular outcomes. METHODS: In a prospective study of 62 patients diagnosed with cancer followed for 24 months from cancer diagnosis through to survivorship (post-cancer treatment), we ascertained baseline fat depots including intermuscular fat (IMF) of the erector spinae muscles; and pre- and post-cancer treatment left ventricular ejection fraction (LVEF) and HF symptoms at baseline and 24-months, respectively. Linear regression was used to model independent variables in relation to HF symptoms at 24-months. RESULTS: Baseline IMF and LVEF change over 24-months significantly interacted to predict HF score at 24-months. The highest HF symptom score was observed for participants who experienced high IMF at baseline and a high decline in LVEF over 24-months (HF score = 11.0) versus all other categories of baseline IMF and LVEF change. CONCLUSIONS: Together IMF and LVEF decline may play an important role in the worsening of HF symptoms in cancer survivors. The finding that IMF at cancer diagnosis led to elevated HF scores post-treatment suggests that IMF may be a potential target for intervention studies.

RNA-seq analyses reveal that cervical spinal cords and anterior motor neurons from amyotrophic lateral sclerosis subjects show reduced expression of mitochondrial DNA-encoded respiratory genes, and rhTFAM may correct this respiratory deficiency.

Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSC's rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.

Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis.

Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non-small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977-89. ©2016 AACR.

Postmortem Alzheimer's Disease Hippocampi Show Oxidative Phosphorylation Gene Expression Opposite that of Isolated Pyramidal Neurons.

Causes of initiation and progression of sporadic Alzheimer's disease (sAD) are likely multiple and include impairment of mitochondrial bioenergetics. We analyzed RNA expression levels of multiple mitochondrial oxidative phosphorylation (OXPHOS) and biogenesis (mitobiogenesis) genes in unfixed hippocampal (WH) frozen sections (10 sAD; 9 CTL) and laser-captured hippocampal pyramidal neurons (PyNs, ~1000 neurons from each case) from 8 sAD and 7 CTL cases. Nuclear-encoded OXPHOS genes in WH were significantly increased in sAD, whereas in isolated sAD PyNs, these same genes were significantly decreased. Mitochondrial DNA-encoded genes were increased in sAD PyNs but showed a non-significant downward trend in sAD WH. Relationships among WH and PyN gene expression levels in sAD distributed in a different population compared to CTL. Principal component analysis (PCA) revealed clustering of CTL but widespread heterogeneity of sAD samples. In sAD, mitochondrial bioenergetics at the gene expression level are depressed in vulnerable PyNs. PCA revealed that CTL samples clustered together, whereas sAD samples varied widely. From the perspective of OXPHOS bioenergetics, sAD is a heterogeneous syndrome and not likely due to a single abnormality. Increased stimulation of nuclear-encoded OXPHOS gene expression in PyNs is a rational therapeutic approach for most but not all cases of sAD.

Mitochondrial oxidative phosphorylation transcriptome alterations in human amyotrophic lateral sclerosis spinal cord and blood.

Origins of onset and progression of motor neurodegeneration in amyotrophic lateral sclerosis (ALS) are not clearly known, but may include impairment of mitochondrial bioenergetics. We used quantitative PCR approaches to analyze the mitochondrial oxidative phosphorylation (OXPHOS) transcriptomes of spinal cord tissue and peripheral blood mononuclear cells (PBMC) from persons with sporadic ALS compared with those without neurological disease. Expression measurements of 88 different nuclear (n) and mitochondrial (mt) DNA-encoded OXPHOS genes showed mtDNA-encoded respiratory gene expression was significantly decreased in ALS spinal cord by 78-84% (ANOVA p < 0.002). We observed the same phenomenon in freshly isolated PBMC from ALS patients (reduced 24-35%, ANOVA p < 0.001) and reproduced it in a human neural stem cell model treated with 2',3'-dideoxycytidine (ddC) (reduced 52-78%, ANOVA p < 0.001). nDNA-encoded OXPHOS genes showed heterogeneously and mostly decreased expression in ALS spinal cord tissue. In contrast, ALS PBMC and ddC-treated stem cells showed no significant change in expression of nDNA OXPHOS genes compared with controls. Genes related to mitochondrial biogenesis (PGC-1α, TFAM, ERRα, NRF1, NRF2 and POLG) were queried with inconclusive results. Here, we demonstrate there is a systemic decrease in mtDNA gene expression in ALS central and peripheral tissues that support pursuit of bioenergetic-enhancing therapies. We also identified a combined nDNA and mtDNA gene set (n = 26), downregulated in spinal cord tissue that may be useful as a biomarker in the development of cell-based ALS models.

Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aß1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aß25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

Preservation of fine-needle aspiration specimens for future use in RNA-based molecular testing.

BACKGROUND: The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues. METHODS: FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif). RESULTS: Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas. CONCLUSIONS: FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies.

Genes involved in radiation therapy response in head and neck cancers.

OBJECTIVES: This is a pilot study designed to identify gene expression profiles able to stratify head and neck squamous cell carcinoma (HNSCC) tumors that may or may not respond to chemoradiation or radiation therapy. STUDY DESIGN: We prospectively evaluated 14 HNSCC specimens, arising from patients undergoing chemoradiotherapy or radiotherapy alone with curative intent. A complete response was assessed by clinical evaluation with no evidence of gross tumor after a 2-year follow-up period. METHODS: Residual biopsy samples from eight complete responders (CR) and six nonresponders (NR) were evaluated by genome-wide gene expression profiling using HG-U133A 2.0 arrays. Univariate parametric t-tests with proportion of false discoveries controlled by multivariate permutation tests were used to identify genes with significantly different gene expression levels between CR and NR cases. Six different prediction algorithms were used to build gene predictor lists. Three representative genes showing 100% crossvalidation support after leave-one-out crossvalidation (LOOCV) were further validated using real-time QRT-PCR. RESULTS: We identified 167 significant probe sets that discriminate between the two classes, which were used to build gene predictor lists. Thus, 142 probe sets showed an accuracy of prediction ranging from 93% to 100% across all six prediction algorithms. The genes represented by these 142 probe sets were further classified into different functional networks that included cellular development, cellular movement, and cancer. CONCLUSIONS: The results presented herein offer encouraging preliminary data that may provide a basis for a more precise prognosis of HNSCC, as well as a molecular-based therapy decision for the management of these cancers.

Assessing the impact of tissue devitalization time on genome-wide gene expression analysis in ovarian tumor samples.

The utilization of genome-wide gene expression microarray technology in tumor stratification has proven a powerful tool to identify gene expression signatures associated with cancer prognosis and is currently under evaluation in clinical laboratories. Standardized protocols, including tumor tissue postoperatively handling guidelines are yet to be defined. We aimed at assessing a systematic effect of devitalization in ovarian tumors' gene expression profiling, using high-density oligonucleotide microarrays, under a standardized protocol following strict quality control criteria. Residual tissue from the surgical pathology specimen was divided into 5 samples. Half of each was immediately snap frozen in liquid nitrogen. The remaining halves were kept at room temperature for 0, 15, 30, 60, and 120 minutes, at which time the tissue was snap frozen in liquid nitrogen, and stored at -80 degrees C until RNA extraction. The entire process from RNA extraction through feature intensity distribution was rigorously monitored for quality. Identification of altered gene expression among each pair of snap frozen and devitalized samples per ovarian tumor specimen was assessed by using the Significance score (S-score) method. We identified only 4 probe sets that seemed to correlate with devitalization time in one of the ovarian tumor specimens, suggesting that they are not likely to have an impact on gene expression profiling tumor stratification. Our study suggests that with proper sample handling and rigorous quality control procedures for RNA extraction and microarray analysis, tumor classification based on global gene expression data will not be adversely affected if devitalization times are kept within a 120-minute window.

Evaluation of quality-control criteria for microarray gene expression analysis.

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.