Human serum albumin-based drug-free macromolecular therapeutics induce apoptosis in chronic lymphocytic leukemia patient cells by crosslinking of CD20 and/or CD38 receptors.

This study explores the efficacy of human serum albumin (HSA)-based Drug-Free Macromolecular Therapeutics (DFMT) in treating Chronic Lymphocytic Leukemia (CLL), a prevalent adult leukemia subtype. DFMT, a novel strategy, employs biomimetic crosslinking of CD20 and CD38 receptors on malignant B cells without the need for low molecular weight drugs. Apoptosis is initiated via a two-step process: i) Recognition of a bispecific engager, Fab' fragment conjugated with morpholino oligonucleotide (Fab'-MORF1), by a cell surface antigen; followed by ii) crosslinking of the MORF1-decorated cells with a multivalent effector, HSA holding multiple copies of complementary MORF2, HSA-(MORF2)x. Herein we evaluated the efficacy of HSA-based DFMT in the treatment of 56 samples isolated from patients diagnosed with CLL. Fab' fragments from Obinutuzumab (OBN) and Isatuximab (ISA) were employed in the synthesis of anti-CD20 (Fab'OBN-MORF1) and anti-CD38 (Fab'ISA-MORF1) bispecific engagers. The efficacy of DFMT was significantly influenced by the expression levels of CD20 and CD38 receptors. Dual-targeting DFMT strategies (CD20 + CD38) were more effective than single-target approaches, particularly in samples with elevated receptor expression. Pretreatment of patient cells with gemcitabine or ricolinostat markedly increased cell surface CD20 and CD38 expression, respectively. Apoptosis was effectively initiated in 62.5% of CD20-targeted samples and in 42.9% of CD38-targeted samples. Our findings demonstrate DFMT's potential in personalized CLL therapy. Further research is needed to validate these outcomes in a larger number of patient samples and to explore DFMT's applicability to other malignancies.

Elevated IgM and abnormal free light chain ratio are increased in relatives from high-risk chronic lymphocytic leukemia pedigrees.

Abnormal serum immunoglobulin (Ig) free light chains (FLC) are established biomarkers of early disease in multiple B-cell lymphoid malignancies, including chronic lymphocytic leukemia (CLL). Heavy chains have also been shown to be biomarkers in plasma cell disorders. An unanswered question is whether these Ig biomarkers are heritable, i.e., influenced by germline factors. CLL is heritable but highly heterogeneous. Heritable biomarkers could elucidate steps of disease pathogenesis that are affected by germline factors, and may help partition heterogeneity and identify genetic pleiotropies across malignancies. Relatives in CLL pedigrees present an opportunity to identify heritable biomarkers. We compared FLCs and heavy chains between relatives in 23 high-risk CLL pedigrees and population controls. Elevated IgM (eIgM) and abnormal FLC (aFLC) ratio was significantly increased in relatives, suggesting that these Ig biomarkers are heritable and could offer risk stratification in pedigree relatives. Within high-risk CLL pedigrees, B-cell lymphoid malignancies were five times more prevalent in close relatives of individuals with eIgM, prostate cancer was three times more prevalent in relatives of individuals with aFLC, and monoclonal B-cell lymphocytosis increased surrounding individuals with normal Ig levels. These different clustering patterns suggest Ig biomarkers have the potential to partition genetic heterogeneity in CLL and provide insight into distinct heritable pleiotropies associated with CLL.

Reparameterization of PAM50 Expression Identifies Novel Breast Tumor Dimensions and Leads to Discovery of a Genome-Wide Significant Breast Cancer Locus at 12q15.

Background: Breast tumor subtyping has failed to provide impact in susceptibility genetics. The PAM50 assay categorizes breast tumors into: Luminal A, Luminal B, HER2-enriched and Basal-like. However, tumors are often more complex than simple categorization can describe. The identification of heritable tumor characteristics has potential to decrease heterogeneity and increase power for gene finding.Methods: We used 911 sporadic breast tumors with PAM50 expression data to derive tumor dimensions using principal components (PC). Dimensions in 238 tumors from high-risk pedigrees were compared with the sporadic tumors. Proof-of-concept gene mapping, informed by tumor dimension, was performed using Shared Genomic Segment (SGS) analysis.Results: Five dimensions (PC1-5) explained the majority of the PAM50 expression variance: three captured intrinsic subtype, two were novel (PC3, PC5). All five replicated in 745 TCGA tumors. Both novel dimensions were significantly enriched in the high-risk pedigrees (intrinsic subtypes were not). SGS gene-mapping in a pedigree identified a 0.5 Mb genome-wide significant region at 12q15 This region segregated through 32 meioses to 8 breast cancer cases with extreme PC3 tumors (P = 2.6 × 10-8).Conclusions: PC analysis of PAM50 gene expression revealed multiple independent, quantitative measures of tumor diversity. These tumor dimensions show evidence for heritability and potential as powerful traits for gene mapping.Impact: Our study suggests a new approach to describe tumor expression diversity, provides new avenues for germline studies, and proposes a new breast cancer locus. Similar reparameterization of expression patterns may inform other studies attempting to model the effects of tumor heterogeneity. Cancer Epidemiol Biomarkers Prev; 27(6); 644-52. ©2018 AACR.

Patency of the Viabahn stent graft for the treatment of outflow stenosis in hemodialysis grafts.

BACKGROUND: To evaluate arteriovenous graft patency when failing grafts are treated with Viabahn covered stents vs percutaneous angioplasty (PTA) alone. METHODS: A retrospective review of all patients that underwent endovascular interventions for failing grafts at a single institution between January 2010 and July 2013 was performed. Forty-four patients were identified who were treated with PTA alone (11) and with Viabahn stent grafts (33) for stenoses in the venous to graft anastomoses. Patient demographics, procedural success, and intraoperative complications were recorded as well as graft patency at 3, 6, and 12 months. Graft patency was reviewed and compared with PTA alone. RESULTS: There was no statistically significant difference between the 2 groups regarding gender, frequency of diabetes, hypertension, coronary artery disease, or peripheral arterial disease. Primary technical success defined as residual stenosis 10% or less was achieved in 100% of the cases. Follow-up was determined by flow velocities during dialysis and ultrasound imaging in the vascular laboratory. At 12 months 87.8% (29/33) grafts with stents were functional vs 36.4% (4/11) of those with PTA alone. Primary patency of the stent group was 61%, 52%, and 42% at 3, 6, and 12 months respectively vs the PTA group 64%, 45%, and 9%. CONCLUSIONS: Grafts treated with Viabahn covered stents for outflow stenosis have a superior patency to PTA alone, 12 months after treatment; although earlier post treatment results are comparable.

Discordant Haplotype Sequencing Identifies Functional Variants at the 2q33 Breast Cancer Risk Locus.

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916-25. ©2016 AACR.

Genome-wide association study identifies variants at 16p13 associated with survival in multiple myeloma patients.

Here we perform the first genome-wide association study (GWAS) of multiple myeloma (MM) survival. In a meta-analysis of 306 MM patients treated at UCSF and 239 patients treated at the Mayo clinic, we find a significant association between SNPs near the gene FOPNL on chromosome 16p13 and survival (rs72773978; P=6 × 10(-10)). Patients with the minor allele are at increased risk for mortality (HR: 2.65; 95% CI: 1.94-3.58) relative to patients homozygous for the major allele. We replicate the association in the IMMEnSE cohort including 772 patients, and a University of Utah cohort including 318 patients (rs72773978 P=0.044). Using publicly available data, we find that the minor allele was associated with increased expression of FOPNL and increased expression of FOPNL was associated with higher expression of centrosomal genes and with shorter survival. Polymorphisms at the FOPNL locus are associated with survival among MM patients.

Genome-wide association study identifies multiple risk loci for chronic lymphocytic leukemia.

Genome-wide association studies (GWAS) have previously identified 13 loci associated with risk of chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL). To identify additional CLL susceptibility loci, we conducted the largest meta-analysis for CLL thus far, including four GWAS with a total of 3,100 individuals with CLL (cases) and 7,667 controls. In the meta-analysis, we identified ten independent associated SNPs in nine new loci at 10q23.31 (ACTA2 or FAS (ACTA2/FAS), P=1.22×10(-14)), 18q21.33 (BCL2, P=7.76×10(-11)), 11p15.5 (C11orf21, P=2.15×10(-10)), 4q25 (LEF1, P=4.24×10(-10)), 2q33.1 (CASP10 or CASP8 (CASP10/CASP8), P=2.50×10(-9)), 9p21.3 (CDKN2B-AS1, P=1.27×10(-8)), 18q21.32 (PMAIP1, P=2.51×10(-8)), 15q15.1 (BMF, P=2.71×10(-10)) and 2p22.2 (QPCT, P=1.68×10(-8)), as well as an independent signal at an established locus (2q13, ACOXL, P=2.08×10(-18)). We also found evidence for two additional promising loci below genome-wide significance at 8q22.3 (ODF1, P=5.40×10(-8)) and 5p15.33 (TERT, P=1.92×10(-7)). Although further studies are required, the proximity of several of these loci to genes involved in apoptosis suggests a plausible underlying biological mechanism.

Deep sequencing for the detection of virus-like sequences in the brains of patients with multiple sclerosis: detection of GBV-C in human brain.

Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1-2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a "hit". Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4-10 million sequences ("reads") each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.

Different mechanisms preserve translation of programmed cell death 8 and JunB in virus-infected endothelial cells.

OBJECTIVE: Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown. METHODS AND RESULTS: We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with polysomes, and 0.6% were enriched ≥2-fold in polysome fractions from infected ECs. Quantitative reverse transcription-polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein expression in PV-infected ECs. The presence of IRESs in the 5' untranslated region of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis. CONCLUSIONS: We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.

C21orf91 genotypes correlate with herpes simplex labialis (cold sore) frequency: description of a cold sore susceptibility gene.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) infects >70% of the United States population. We identified a 3-megabase region on human chromosome 21 containing 6 candidate genes associated with herpes simplex labialis (HSL, "cold sores"). METHODS: We conducted single nucleotide polymorphism (SNP) scans of the chromosome 21 region to define which of 6 possible candidate genes were associated with cold sore frequency. We obtained the annual HSL frequency for 355 HSV-1 seropositive individuals and determined the individual genotypes by SNPlex for linkage analysis and parental transmission disequilibrium testing (ParenTDT). RESULTS: Two-point linkage analysis showed positive linkage between cold sore frequency and 2 SNPs within the C21orf91 region, 1 of which is nonsynonymous. ParenTDT analysis revealed a strong association between another C21orf91 SNP, predicted to lie in the 3' untranslated region, and frequent HSL (P = .0047). C21orf 91 is a predicted open reading frame of unknown function that encodes a cytosolic protein. CONCLUSIONS: We evaluated candidate genes in the cold sore susceptibility region using fine mapping with 45 SNP markers. 2 complementary techniques identified C21orf91 as a gene of interest for susceptibility to HSL. We propose that C21orf91 be designated the Cold Sore Susceptibility Gene-1 (CSSG1).

Scanning electron microscopy visualization of methicillin-resistant Staphylococcus aureus after contact with gatifloxacin with and without preservative.

PURPOSE: To test a visual model by looking at the differences in effect of Zymar((R)) (gatifloxacin plus benzalkonium chloride [BAK]) when compared to gatifloxacin and a normal saline (NS) control upon a methicillin and gatifloxacin-resistant Staphylococcus aureus (MRSA) species. METHODS: An ocular isolate of gatifloxacin-resistant (minimal inhibitory concentration >2 to 4 microg/mL) MRSA was grown to confluency. Chambered slides were prepared with bacterial culture smears, and then incubated with either gatifloxacin at the concentrations of 1 and 10 microg/mL, Zymar containing equivalent concentrations of gatifloxacin, or NS. Bacterial cultures were fixed after 10, 30, and 60 min. Fixed slides were coated in gold sputter for examination. Bacteria were visually evaluated with scanning electron microscopy (SEM) at 50,000x. Blinded review of SEM images compared structural changes and mitotic activity across samples. RESULTS: MRSA exposed to 10 microg Zymar for 60 min showed significantly greater pleomorphism and cell wall surface changes when compared to gatifloxacin (P < 0.0001) and NS (P = 0.001), and significantly less mitotic activity than NS (P = 0.002). CONCLUSION: Using SEM, the topical formulation of gatifloxacin 0.3% (Zymar), which contains BAK, had greater antibacterial activity than did gatifloxacin alone in gatifloxacin and methicillin-resistant S. aureus, thereby illustrating potential advantages of the preservative in the commercial formulation. We further show that these effects can be visualized and quantified.

Identification of a herpes simplex labialis susceptibility region on human chromosome 21.

BACKGROUND: Most of the United States population is infected with either herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, or both. Reactivations of HSV-1 infection cause herpes simplex labialis (HSL; cold sores or fever blisters), which is the most common recurring viral infection in humans. METHODS: To investigate the possibility of a human genetic component conferring resistance or susceptibility to cold sores (i.e., a HSL susceptibility gene), we conducted a genetic linkage analysis that included serotyping and phenotyping 421 individuals from 39 families enrolled in the Utah Genetic Reference Project. RESULTS: Linkage analysis identified a 2.5-Mb nonrecombinant region of interest on the long arm of human chromosome 21, with a multipoint logarithm of odds score of 3.9 noted near marker abmc65 (D21S409). Nonparametric linkage analysis of the data also provided strong evidence for linkage (P = .0005). This region of human chromosome 21 contains 6 candidate genes for herpes susceptibility. CONCLUSIONS: The development of frequent cold sores is associated with a region on the long arm of human chromosome 21. This region contains several candidate genes that could influence the frequency of outbreaks of HSL.

STAT1 binds to the herpes simplex virus type 1 latency-associated transcript promoter.

The authors hypothesized that environmental stimuli induce cytokines that act through an intracellular cascade, which includes signal transducers and activators of transcription (STATs), to change herpes simplex virus (HSV) gene expression, thereby inducing viral reactivation. The HSV type 1 (HSV-1) latency-associated transcript (LAT) gene regulates viral reactivation within neurons via an unknown mechanism. HSV-1 deletion mutants that are missing key portions of the LAT gene, particularly the 3' region of the LAT promoter, do not reactivate normally in vivo. The authors hypothesized that STAT transcription factors may bind in this region to regulate viral reactivation. Electrophoretic mobility shift assay (EMSA) experiments were performed by incubating mouse trigeminal ganglion (TG) nuclear extracts with each of three overlapping sequences representing the 3' region of the HSV-1 LAT promoter (referred to as oligos L1, L2, and L3). The ganglionic nuclear extracts bound specifically to oligos L1 and L3, but not L2. Oligos L1 and L3 contain predicted STAT binding sequences whereas L2 does not. Specific binding to oligo L3 (including the TATA box sequence) was supershifted by incubating with anti-STAT1 antibodies, but not by incubating with anti-STAT3 or anti-STAT5a antibodies. Specific L3 binding was reduced by competing with excess unlabeled STAT1 consensus sequences. These results indicate that STAT1, probably as part of a complex, is capable of binding to the LAT promoter on or near the TATA box. Further studies are required to determine if STAT1 is required for LAT expression in vivo. This work supports the hypothesis that interferons act through STAT1 to regulate the expression of HSV-1 LAT.