Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses.

The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.

Apo CIII Proteoforms, Plasma Lipids, and Cardiovascular Risk in MESA.

BACKGROUND: Apo CIII (apolipoprotein CIII) is an important regulator of triglyceride metabolism and was associated with cardiovascular risk in several cohorts. It is present in 4 major proteoforms, a native peptide (CIII0a), and glycosylated proteoforms with zero (CIII0b), 1 (CIII1, most abundant), or 2 (CIII2) sialic acids, which may differentially modify lipoprotein metabolism. We studied the relationships of these proteoforms with plasma lipids and cardiovascular risk. METHODS: Apo CIII proteoforms were measured by mass spectrometry immunoassay in baseline plasma samples of 5791 participants of Multi-Ethnic Study of Atherosclerosis, an observational community-based cohort. Standard plasma lipids were collected for up to 16 years and cardiovascular events (myocardial infarction, resuscitated cardiac arrest, or stroke) were adjudicated for up to 17 years. RESULTS: Apo CIII proteoform composition differed by age, sex, race and ethnicity, body mass index, and fasting glucose. Notably, CIII1 was lower in older participants, men and Black and Chinese (versus White) participants, and higher in obesity and diabetes. In contrast, CIII2 was higher in older participants, men, Black, and Chinese persons, and lower in Hispanic individuals and obesity. Higher CIII2 to CIII1 ratio (CIII2/III1) was associated with lower triglycerides and higher HDL (high-density lipoprotein) in cross-sectional and longitudinal models, independently of clinical and demographic risk factors and total apo CIII. The associations of CIII0a/III1 and CIII0b/III1 with plasma lipids were weaker and varied through cross-sectional and longitudinal analyses. Total apo CIII and CIII2/III1 were positively associated with cardiovascular disease risk (n=669 events, hazard ratios, 1.14 [95% CI, 1.04-1.25] and 1.21 [1.11-1.31], respectively); however, the associations were attenuated after adjustment for clinical and demographic characteristics (1.07 [0.98-1.16]; 1.07 [0.97-1.17]). In contrast, CIII0b/III1 was inversely associated with cardiovascular disease risk even after full adjustment including plasma lipids (0.86 [0.79-0.93]). CONCLUSIONS: Our data indicate differences in clinical and demographic relationships of apo CIII proteoforms, and highlight the importance of apo CIII proteoform composition in predicting future lipid patterns and cardiovascular disease risk.

Renal Transporter Alterations in Patients with Chronic Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination.

Alterations in hepatic transporters have been identified in precirrhotic chronic liver diseases (CLDs) that result in pharmacokinetic variations causing adverse drug reactions (ADRs). However, the effect of CLD on the expression of renal transporters is unknown despite the overwhelming evidence of kidney injury in CLD patients. This study determines the transcriptomic and proteomic expression profiles of renal drug transporters in patients with defined CLD etiology. Renal biopsies were obtained from patients with a history of CLD etiologies, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), viral hepatitis C (HCV), and combination ALD/HCV. A significant decrease in organic anion transporter (OAT)-3 was identified in NASH, ALD, HCV, and ALD/HCV (1.56 ± 1.10; 1.01 ± 0.46; 1.03 ± 0.43; 0.86 ± 0.57 pmol/mg protein) relative to control (2.77 ± 1.39 pmol/mg protein). Additionally, a decrease was shown for OAT4 in NASH (24.9 ± 5.69 pmol/mg protein) relative to control (43.8 ± 19.9 pmol/mg protein) and in urate transporter 1 (URAT1) for ALD and HCV (1.56 ± 0.15 and 1.65 ± 0.69 pmol/mg protein) relative to control (4.69 ± 4.59 pmol/mg protein). These decreases in organic anion transporter expression could result in increased and prolonged systemic exposure to drugs and possible toxicity. Renal transporter changes, in addition to hepatic transporter alterations, should be considered in dose adjustments for CLD patients for a more accurate disposition profile. It is important to consider a multiorgan approach to altered pharmacokinetics of drugs prescribed to CLD patients to prevent ADRs and improve patient outcomes. SIGNIFICANCE STATEMENT: Chronic liver diseases are known to elicit alterations in hepatic transporters that result in a disrupted pharmacokinetic profile for various drugs. However, it is unknown if there are alterations in renal transporters during chronic liver disease, despite strong indications of renal dysfunction associated with chronic liver disease. Identifying renal transporter expression changes in patients with chronic liver disease facilitates essential investigations on the multifaceted relationship between liver dysfunction and kidney physiology to offer dose adjustments and prevent adverse drug reactions.

Plasma proteoforms of apolipoproteins C-I and C-II are associated with plasma lipids in the Multi-Ethnic Study of Atherosclerosis.

Apolipoproteins (apo) C-I and C-II are key regulators of triglyceride and HDL metabolism. Both exist as full-size native and truncated (apoC-I'; apoC-II') posttranslational proteoforms. However, the determinants and the role of these proteoforms in lipid metabolism are unknown. Here, we measured apoC-I and apoC-II proteoforms by mass spectrometry immunoassay in baseline and 10-year follow-up plasma samples from the Multi-Ethnic Study of Atherosclerosis. We found that baseline total apoC-I (mean = 9.2 mg/dl) was lower in African Americans (AA), Chinese Americans (CA), and Hispanics (by 1.8; 1.0; 1.0 mg/dl vs. whites), higher in women (by 1.2 mg/dl), and positively associated with plasma triglycerides and HDL. Furthermore, we observed that the truncated-to-native apoC-I ratio (apoC-I'/C-I) was lower in CA, negatively associated with triglycerides, and positively associated with HDL. We determined that total apoC-II (8.8 mg/dl) was lower in AA (by 0.8 mg/dl) and higher in CA and Hispanics (by 0.5 and 0.4 mg/dl), positively associated with triglycerides, and negatively associated with HDL. In addition, apoC-II'/C-II was higher in AA and women, negatively associated with triglycerides, and positively associated with HDL. We showed that the change in triglycerides was positively associated with changes in total apoC-I and apoC-II and negatively associated with changes in apoC-I'/C-I and apoC-II'/C-II, whereas the change in HDL was positively associated with changes in total apoC-I and apoC-II'/C-II and negatively associated with change in total apoC-II. This study documents racial/ethnic variation in apoC-I and apoC-II plasma levels and highlights apolipoprotein posttranslational modification as a potential regulator of plasma lipids.

Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes.

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.

Germline mutations in HOXB13 and prostate-cancer risk.

BACKGROUND: Family history is a significant risk factor for prostate cancer, although the molecular basis for this association is poorly understood. Linkage studies have implicated chromosome 17q21-22 as a possible location of a prostate-cancer susceptibility gene. METHODS: We screened more than 200 genes in the 17q21-22 region by sequencing germline DNA from 94 unrelated patients with prostate cancer from families selected for linkage to the candidate region. We tested family members, additional case subjects, and control subjects to characterize the frequency of the identified mutations. RESULTS: Probands from four families were discovered to have a rare but recurrent mutation (G84E) in HOXB13 (rs138213197), a homeobox transcription factor gene that is important in prostate development. All 18 men with prostate cancer and available DNA in these four families carried the mutation. The carrier rate of the G84E mutation was increased by a factor of approximately 20 in 5083 unrelated subjects of European descent who had prostate cancer, with the mutation found in 72 subjects (1.4%), as compared with 1 in 1401 control subjects (0.1%) (P=8.5x10(-7)). The mutation was significantly more common in men with early-onset, familial prostate cancer (3.1%) than in those with late-onset, nonfamilial prostate cancer (0.6%) (P=2.0x10(-6)). CONCLUSIONS: The novel HOXB13 G84E variant is associated with a significantly increased risk of hereditary prostate cancer. Although the variant accounts for a small fraction of all prostate cancers, this finding has implications for prostate-cancer risk assessment and may provide new mechanistic insights into this common cancer. (Funded by the National Institutes of Health and others.).

Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors.

Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer.

Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides.

The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments.

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.

Preclinical investigation of nanoparticle albumin-bound paclitaxel as a potential treatment for adrenocortical cancer.

BACKGROUND: Traditional drug discovery methods have a limited role in rare cancers. We hypothesized that molecular technology including gene expression profiling could expose novel targets for therapy in adrenocortical carcinoma (ACC), a rare and lethal cancer. SPARC (secreted protein acidic rich in cysteine) is an albumin-binding matrix-associated protein that is proposed to act as a mechanism for the increased efficacy of a nanoparticle albumin-bound preparation of the antimicrotubular drug Paclitaxel (nab-paclitaxel). METHODS: The transcriptomes of 19 ACC tumors and 4 normal adrenal glands were profiled on Affymetrix U133 Plus2 expression microarrays to identify genes representing potential therapeutic targets. Immunohistochemical analysis for target proteins was performed on 10 ACC, 6 benign adenomas, and 1 normal adrenal gland. Agents known to inhibit selected targets were tested in comparison with mitotane in the 2 ACC cell lines (H295R and SW-13) in vitro and in mouse xenografts. RESULTS: SPARC expression is increased in ACC samples by 1.56 ± 0.44 (µ ± SD) fold. Paclitaxel and nab-paclitaxel show in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 µM and 0.0078 µM for paclitaxel and 0.35 µM and 0.0087 µM for nab-paclitaxel compared with mitotane concentrations of 15.9 µM and 46.4 µM. In vivo nab-paclitaxel treatment shows a greater decrease in tumor weight in both xenograft models than mitotane. CONCLUSIONS: Biological insights garnered through expression profiling of ACC tumors suggest further investigation into the use of nab-paclitaxel for the treatment of ACC.

Increased Renal Expression of Complement Components in Patients With Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination.

Inflammatory liver diseases, including nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), hepatitis C virus (HCV), and ALD/HCV, account for nearly 2 million deaths annually. Despite increasing evidence that liver dysfunction impacts renal physiology, there is limited supportive clinical information, due to limited diagnosis of liver disease, complexity in liver disease etiology, and inadequacy of renal function tests. Human kidney biopsies with liver and renal pathology were obtained from patients with nonalcoholic fatty liver disease (NAFLD), NASH, ALD, HCV, and ALD/HCV (n = 5-7). Each liver disease showed renal pathology with at least 50% interstitial nephritis, 50% interstitial fibrosis, and renal dysfunction by estimated glomerular filtration rate (NAFLD 36.7 ± 21.4; NASH 32.7 ± 15.0; ALD 16.0 ± 11.0; HCV 27.6 ± 11.5; ALD/HCV 21.0 ± 11.2 ml/min/1.73 m2). Transcriptomic analysis identified 55 genes with expression changes in a conserved direction in response to liver disease. Considering association with immune regulation, protein levels of alpha-2-macroglobulin, clusterin, complement C1q C chain (C1QC), CD163, and joining chain of multimeric IgA and IgM (JCHAIN) were further quantified by LC-MS/MS. C1QC demonstrated an increase in NASH, ALD, HCV, and ALD/HCV (42.9 ± 16.6; 38.8 ± 18.4; 39.0 ± 13.5; 40.1 ± 20.1 pmol/mg protein) relative to control (19.2 ± 10.4 pmol/mg protein; p ≤ 0.08). Renal expression changes identified in inflammatory liver diseases with interstitial pathology suggest the pathogenesis of liver associated renal dysfunction. This unique cohort overcomes diagnostic discrepancies and sample availability to provide insight for mechanistic investigations on the impact of liver dysfunction on renal physiology.

Next-generation sequencing of Coccidioides immitis isolated during cluster investigation.

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source.

Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context. RESULTS: We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation. CONCLUSIONS: We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.

Per- and Polyfluoroalkyl Substance and Cardio Metabolic Markers in Firefighters.

OBJECTIVE: To evaluate if serum polyfluoroalkyl substances (PFAS) were associated with cardiometabolic markers. METHODS: Serum PFAS were evaluated in 38 Arizona firefighters and 49 participants from the 2009 to 2010 National Health and Nutrition Examination Survey (NHANES). Cardiometabolic markers including carotid intima-medial thickness (CIMT) were measured in the firefighters. RESULTS: Firefighters had elevated perfluorohexane sulfonic acid (PFHxS) and lower perfluorononanoic acid (PFNA) and perfluoroundecanoic acid (PFUA) compared to NHANES participants; for nine of the other 12 PFAS the values were not significantly different. There were significant negative associations among firefighters between perfluorodecanoic acid (PFDeA) and total cholesterol and PFUA and interleukin-6. PFAS concentrations were not associated with CIMT. CONCLUSION: PFHxS levels were elevated in firefighters compared to NHANES subjects. Serum PFAS concentrations were not associated with increased cardiometabolic risk measures in this population of firefighters.

Gender Difference in Damage-Mediated Signaling Contributes to Pulmonary Arterial Hypertension.

Aims: Pulmonary arterial hypertension (PAH) is a progressive lethal disease with a known gender dimorphism. Female patients are more susceptible to PAH, whereas male patients have a lower survival rate. Initial pulmonary vascular damage plays an important role in PAH pathogenesis. Therefore, this study aimed at investigating the role of gender in activation of apoptosis/necrosis-mediated signaling pathways in PAH. Results: The media collected from pulmonary artery endothelial cells (PAECs) that died by necrosis or apoptosis were used to treat naive PAECs. Necrotic cell death stimulated phosphorylation of toll-like receptor 4, accumulation of interleukin 1 beta, and expression of E-selectin in a redox-dependent manner; apoptosis did not induce any of these effects. In the animal model of severe PAH, the necrotic marker, high mobility group box 1 (HMGB1), was visualized in the pulmonary vascular wall of male but not female rats. This vascular necrosis was associated with male-specific redox changes in plasma, activation of the same inflammatory signaling pathway seen in response to necrosis in vitro, and an increased endothelial-leukocyte adhesion in small pulmonary arteries. In PAH patients, gender-specific changes in redox homeostasis correlated with the prognostic marker, B-type natriuretic peptide. Males had also shown elevated circulating levels of HMGB1 and pro-inflammatory changes. Innovation: This study discovered the role of gender in the initiation of damage-associated signaling in PAH and highlights the importance of the gender-specific approach in PAH therapy. Conclusion: In PAH, the necrotic cell death is augmented in male patients compared with female patients. Factors released from necrotic cells could alter redox homeostasis and stimulate inflammatory signaling pathways.

Diabetes Mellitus Associates with Increased Right Ventricular Afterload and Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: Diabetes mellitus is associated with left ventricular hypertrophy and dysfunction. Parallel studies have also reported associations between diabetes mellitus and right ventricular dysfunction and reduced survival in patients with pulmonary arterial hypertension. However, the impact of diabetes mellitus on the pulmonary vasculature has not been well characterized. We hypothesized that diabetes mellitus and hyperglycemia could specifically influence right ventricular afterload and remodeling in patients with Group I pulmonary arterial hypertension, providing a link to their known susceptibility to right ventricular dysfunction. METHODS: Using an adjusted model for age, sex, pulmonary vascular resistance, and medication use, associations of fasting blood glucose, glycated hemoglobin, and the presence of diabetes mellitus were evaluated with markers of disease severity in 162 patients with pulmonary arterial hypertension. RESULTS: A surrogate measure of increased pulmonary artery stiffness, elevated pulmonary arterial elastance (P = .012), along with reduced log(pulmonary artery capacitance) (P = .006) were significantly associated with the presence of diabetes mellitus in patients with pulmonary arterial hypertension in a fully adjusted model. Similar associations between pulmonary arterial elastance and capacitance were noted with both fasting blood glucose and glycated hemoglobin. Furthermore, right ventricular wall thickness on echocardiography was greater in pulmonary arterial hypertension patients with diabetes, supporting the link between right ventricular remodeling and diabetes. CONCLUSION: Cumulatively, these data demonstrate that an increase in right ventricular afterload, beyond pulmonary vascular resistance alone, may influence right ventricular remodeling and provide a mechanistic link between the susceptibility to right ventricular dysfunction in patients with both diabetes mellitus and pulmonary arterial hypertension.

Association of Cystatin C Proteoforms with Estimated Glomerular Filtration Rate.

Background: Cystatin C (CysC), a marker for chronic kidney disease, exists as three sequence proteoforms, in addition to the wild-type sequence: one contains hydroxyproline at position 3 (3Pro-OH), the two others have truncated sequences (des-S and des-SSP). Here, we examine correlations between each of these CysC proteoforms and estimated glomerular filtration rate (eGFR), a diagnostic criterion for chronic kidney disease (CKD). Methods: CysC proteoform concentrations were determined from the plasma of 297 diabetes patients at a baseline time point and nine-months later, using a mass spectrometric immunoassay, and were correlated with eGFR calculations. Results: In all samples, 3Pro-OH was the most abundant CysC proteoform, followed by the wild-type proteoform. Least abundant were the truncated CysC proteoforms, des-S and des-SSP, although they demonstrated stronger negative correlation with eGFR than the 3Pro-OH and wild-type proteoforms. The des-SSP truncated proteoform exhibited negative predictive value for eGFR. Conclusions: The truncated CysC proteoforms show potential for clinical and prognostic utility in CKD staging. This could be useful in populations where current methods do not provide satisfactory solutions.

Whole genome sequencing reveals potential targets for therapy in patients with refractory KRAS mutated metastatic colorectal cancer.

BACKGROUND: The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy is poor, with median survival of less than one year. The purpose of this study was to identify candidate therapeutically targetable somatic events in mCRC patient samples by whole genome sequencing (WGS), so as to obtain targeted treatment strategies for individual patients. METHODS: Four patients were recruited, all of whom had received > 2 prior therapy regimens. Percutaneous needle biopsies of metastases were performed with whole blood collection for the extraction of constitutional DNA. One tumor was not included in this study as the quality of tumor tissue was not sufficient for further analysis. WGS was performed using Illumina paired end chemistry on HiSeq2000 sequencing systems, which yielded coverage of greater than 30X for all samples. NGS data were processed and analyzed to detect somatic genomic alterations including point mutations, indels, copy number alterations, translocations and rearrangements. RESULTS: All 3 tumor samples had KRAS mutations, while 2 tumors contained mutations in the APC gene and the PIK3CA gene. Although we did not identify a TCF7L2-VTI1A translocation, we did detect a TCF7L2 mutation in one tumor. Among the other interesting mutated genes was INPPL1, an important gene involved in PI3 kinase signaling. Functional studies demonstrated that inhibition of INPPL1 reduced growth of CRC cells, suggesting that INPPL1 may promote growth in CRC. CONCLUSIONS: Our study further supports potential molecularly defined therapeutic contexts that might provide insights into treatment strategies for refractory mCRC. New insights into the role of INPPL1 in colon tumor cell growth have also been identified. Continued development of appropriate targeted agents towards specific events may be warranted to help improve outcomes in CRC.

PTTG1 overexpression in adrenocortical cancer is associated with poor survival and represents a potential therapeutic target.

BACKGROUND: Adrenocortical carcinoma (ACC) is associated with poor survival rates. The objective of the study was to analyze ACC gene expression profiling data for prognostic biomarkers and therapeutic targets. METHODS: We profiled 44 ACC and 4 normal adrenals on Affymetrix U133 Plus 2 expression microarrays. Pathway and transcriptional enrichment analysis was performed. Protein levels were determined by Western blot. Drug efficacy was assessed against ACC cell lines. Previously published expression datasets were analyzed for validation. RESULTS: Pathway enrichment analysis identified marked dysregulation of cyclin-dependent kinases and mitosis. Overexpression of PTTG1, which encodes securin, a negative regulator of p53, was identified as a marker of poor survival. Median survival for patients with tumors expressing high PTTG1 levels (log2 ratio of PTTG1 to average ß-actin <-3.04) was 1.8 years compared with 9.0 years if tumors expressed lower levels of PTTG1 (P < .0001). Analysis of a previously published dataset confirmed the association of high PTTG1 expression with a poor prognosis. Treatment of 2 ACC cell lines with vorinostat decreased securin levels and inhibited cell growth (median inhibition concentrations of 1.69 µmol/L and 0.891 µmol/L, for SW-13 and H295R, respectively). CONCLUSION: Overexpression of PTTG1 is correlated with poor survival in ACC. PTTG1/securin is a prognostic biomarker and warrants investigation as a therapeutic target.

A pilot study using next-generation sequencing in advanced cancers: feasibility and challenges.

PURPOSE: New anticancer agents that target a single cell surface receptor, up-regulated or amplified gene product, or mutated gene, have met with some success in treating advanced cancers. However, patients' tumors still eventually progress on these therapies. If it were possible to identify a larger number of targetable vulnerabilities in an individual's tumor, multiple targets could be exploited with the use of specific therapeutic agents, thus possibly giving the patient viable therapeutic alternatives. EXPERIMENTAL DESIGN: In this exploratory study, we used next-generation sequencing technologies (NGS) including whole genome sequencing (WGS), and where feasible, whole transcriptome sequencing (WTS) to identify genomic events and associated expression changes in advanced cancer patients. RESULTS: WGS on paired tumor and normal samples from nine advanced cancer patients and WTS on six of these patients' tumors was completed. One patient's treatment was based on targets and pathways identified by NGS and the patient had a short-lived PET/CT response with a significant reduction in his tumor-related pain. To design treatment plans based on information garnered from NGS, several challenges were encountered: NGS reporting delays, communication of results to out-of-state participants and their treating oncologists, and chain of custody handling for fresh biopsy samples for Clinical Laboratory Improvement Amendments (CLIA) target validation. CONCLUSION: While the initial effort was a slower process than anticipated due to a variety of issues, we demonstrate the feasibility of using NGS in advanced cancer patients so that treatments for patients with progressing tumors may be improved.