Citations

Abstract Chromosome 13 deletion (Δ13) is one the most common genetic abnormalities observed in multiple myeloma (MM) and confers a poor prognosis. Studies to identify critical tumor suppressor genes in MM till date have lack adequate resolution, and the molecular phenotype associated with Δ13 has not been established. In this study we seek to establish a molecular profile for Δ13 and to finely map the minimal common region of deletion (CDR) on chromosome 13 by using gene expression profiling (GEP) and array comparative genomic hybridization (aCGH). GEP was performed on RNA from purified plasma cells of 72 newly diagnosed MM and 50 human myeloma cell lines (HMCLs) using the Affymetrix U133A chip and U133plus chip (Affymetrix, Santa Clara, CA) respectively. Patients were assigned TC classes which correlates with underlying genetic subtypes. In addition, aCGH was performed on 79 MM samples (36 with GEP data) and 50 myeloma cell lines (HMCLs; 48 with GEP data) using a platform utilizing 60-mer oligonucleotides (Human Genome CGH 44B Oligo Microarrays, Agilent Technologies), which have a resolution of about 70Kb. Raw data was extracted using the Feature Extraction 8.1 and visualized using CGH Analytics 3.2 (Agilent Technologies). To define a signature that reflects the biological consequences of Δ13 and not its close association with some genetic subtypes of MM (example, t(4;14) and t(14;16)), we selected a training cohort consisting of cases belonging to D1, D2, 11q13 and none TC class such that cases with and without Δ13 are balanced for ploidy and TC classes (n=28). A 152-gene Δ13 signature was identified. Its specificity was confirmed by leave-one-out cross validation using the K-nearest neighbor (KNN) class prediction algorithm (predictive accuracy of 100% in the training cohort). It was subsequently validated in a validation cohort that includes t(4;14) and t(14;16) that were not use to derive this gene signature, maintaining a high predictive accuracy (88%). This gene-set may therefore reflect core transcriptional consequences of Δ13 and is enriched for genes involved in the cell cycle and apoptosis. Using aCGH, 20 patients have Δ13 (mostly whole chromosome) all involving the 13q14–q21 region. Of the 32 HMCLs with Δ13 (whole chromosome or interstitial), 22 have mono-allelic loss of RB1 whereas 4 had bi-allelic loss. Besides RB1, bi-allelic loss of several other genes in the 13q14–q21 region (CYSLTR2, CDADC1, ITM2B, PCDH9) was also observed. As the CDR is large and contains a number of genes, we utilized a set of criteria (bi-allelic loss in HMCLs, within 13q14–q21 CDR, gene expression significantly correlated with copy number, and loss could explain molecular phenotype) to narrow down the potential candidates. Only RB1 and ITM2B fulfilled these criteria. Furthermore, RB protein levels correlated well with mRNA levels and DNA copy suggesting that even in absence of mutations resulting in bi-allelic loss, mono-allelic loss of RB1 could be tumorigenic through a haploinsufficiency mechanism.

Abstract Hyperdiploid MM (H-MM), characterized by recurrent trisomies constitute about 50% of MM, yet very little is known about its pathogenesis and oncogenic mechanisms. Studies in leukemia and solid tumors have shown gene dosage effect of aneuploidy on gene expression. To determine the possible gene dosage effect and deregulated cellular program in H-MM we undertook a gene expression study of CD138-enriched plasma-cell RNA from 53 hyperdiploid and 37 non-hyperdiploid MM (NH-MM) patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Gene expression data was analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Genes differentially expressed between H-MM and NH-MM were obtained by t-test (p<0.01). The majority of the differentially expressed genes (57%) were under-expressed in H-MM. Genes located on the commonly trisomic chromosomes were mostly (but not always) over-expressed in H-MM and constitute 76% of over-expressed genes. Chromosome 1 contained the most differentially expressed genes (17%) followed by chromosome 12 (9%), and 19 (8%). To examine the relationship of gene copy number to gene expression, we examined the expression of genes on chromosomes 9 and 15 in subjects with 2 copies (15 normal control and 20 NH-MM) and 3 copies (12 H-MM) of each chromosome as detected by interphase FISH. We then derived a ratio of the mean expression of each gene on these chromosomes between patients with 3 copies and 2 copies of the chromosome. If a simple relationship exists between gene expression and gene copy number, one would expect the ratio of expression of most genes on these two chromosomes to be about 1.5 in H-MM compared to NH-MM. However, many genes have ratios either higher than 2 or lower than 0.5. Furthermore, when the heterogeneity of cells with underlying trisomies is taken into consideration by correcting the ratio for the number of cells with trisomies, the actual ratio is always lower than the expected ratio. When the expression of genes on a chromosome was compressed to a median value, this value was always higher in the trisomic chromosomes for H-MM compared to NH-MM. The data suggests that although gene dosage influence gene expression, the relationship is complex and some genes are more gene dosage dependent than others. Amongst the differentially expressed genes with known function, 33% are involved in mRNA translation/protein synthesis. Of note, 37 of the top 100 differentially expressed genes are involved in these processes. In particular, 60 ribosomal protein (RP) genes are significantly (p<0.05) upregulated in H-MM. This signature in H-MM is not associated with increase proliferation as measured by PCLI. This predominant signature suggests that deregulated protein synthesis may be important for the biology of H-MM. Many of these RPs are involved in the synthesis of product of oncogenic pathways (e.g. MYC, NF-KB pathways) and may mediate the growth and survival of tumor cells. It is therefore possible that these tumor cells may be sensitive to the disruption of mRNA translation/protein synthesis. Targeting the mTOR pathway with rapamycin may therefore be useful for treatment of H-MM.

Abstract Background: Conventional cytogenetic studies in myeloproliferative neoplasms (MPNs) have provided pathogenetic, diagnostic and prognostic information and in certain instances guide therapeutic decisions. It is therefore reasonable to hypothesize that a higher resolution examination of chromosomes might reveal additional changes that are scientifically as well as clinically relevant. Methods: Granulocyte DNA from 70 patients (median age 56 years, range 18–84; 43 males) with classic BCR-ABL-negative MPNs were studied: 27 with primary myelofibrosis (PMF), 22 with polycythemia vera (PV), 12 with essential thrombocythemia (ET), 6 with post-ET MF and 3 with post-PV MF. Twelve healthy volunteers were used as controls. Cytogenetic information, collected at variable times before the current study, was available in 57 patients. Samples were analyzed by comparative genomic hybridization (CGH) with a gender matched normal control using oligoarrays with either 44,000 or 105,000 oligonucleotides. Data were analyzed with CGH Analytics (Agilent). Copy number changes were considered significant (CNCs) if they were defined by 4 or more adjacent oligonucleotides spanning at least 150,000 base pairs, contained at least one gene, and were not identified in the Database of Genomic Variants. Results: Array CGH results were equivocal in 6 cases, which were therefore excluded from further analysis. Among the 12 normal controls, granulocyte DNA displayed no CNCs. The percentage of patients with CNCs was 52% for PMF, 32% for PV and 17% for ET. In addition, 2 of 6 patients with post-ET MF and 1 of 2 patients with post-PV MF displayed CNCs. There was no correlation between the occurrence of CNCs and JAK2V617F mutational status in PMF and ET. Array CGH revealed CNCs in 16 of 49 patients (33%) with normal and 7 of 8 patients (88%) with abnormal cytogenetic findings; one patient with t(2;12)(p13;q14) in 19 of 20 metaphases detected at 9 months prior to sample collection for the array CGH study displayed no CNCs. In patients with abnormal cytogenetic findings, array CGH also detected imbalances at chromosomal translocations, including t(1;6), t(9;11), and t(1;18), as well as additional abnormalities that were not detected by cytogenetic analysis. A total of 23 single occurrence CNCs were documented and included all the ET [del(2)(q31.3q32.1), del(10)(q11.21q11.22)] and post-ET MF [del(2p12), del(Xq12)] associated CNCs. In contrast, several specific abnormalities were shared between PV and PMF including del(6p22) and dup(15q11.2). As expected, del(13q) and del(20q) abnormalities were recurrent in PMF. Conclusions: Conventional cytogenetic analysis underestimates the extent of genomic gains and losses in BCR-ABL-negative MPNs. The current study suggests that oligonucleotide array CGH is more sensitive to detect small genomic alterations and we are currently studying higher resolution arrays in a larger group of patients to detect additional genomic alterations as well as assess overall clinical utility. The current data also suggest that chromosomal translocations are commonly associated with imbalances.

Abstract Introduction: The analysis of what goes awry in a malignant cell has focused primarily on mutations of protein-encoding genes and their regulatory sequences. However, recent work on microRNA (miRNA) has shed light on the possible involvement of miRNA genes in human disease. miRNA are small “noncoding” or “non-messenger” RNAs of about 21–25 nucleotides in length that function as regulators of gene expression essentially by pairing to the mRNA of protein-coding genes to initiate mRNA degradation or repression of translation. We are interested in characterizing miRNA expression profiles in human myeloma cell lines (HMCLs) and myeloma patient samples. Multiple myeloma (MM) is a plasma cell tumor characterized by frequent chromosomal translocations. Materials and methods: The expression of miRNA in HMCLs, primary MM cells (hyperdiploid samples) and normal plasma cells was determined using human microRNA chips. The GenoExporerTM Human microRNA chips were developed by GenoSensor (Tempe, AZ) and the microarray contains 226 human microRNA sequences in addition to control sequences. The DNA oligo probes are synthesized and immobilized on the chips (1”x 3” standard glass microslide). The probes are designed based on the active mature miRNA sequences and some of their flanking sequences. Our analysis included seven myeloma patients (hyperdiploid MM), eight different HMCLs (MM1, 8226 with t(14;16); SKMM2, U266, INA-6 with t(11;14) and H929, KMS11, UTMC-2 with t(4;14)), and eight normal human plasma cells. The normal human plasma cells were collected at Mayo Clinic Rochester from bone samples of patients undergoing orthopedic surgery. For the assay 5–10 μg of total RNA per sample were used at a concentration of 1 μg/μL. The RNA is directly labeled with biotin and used as a target for the on-chip hybridization assays. A streptavidin-Alexa dye is used to stain the hybridized targets and the fluorescent signals are captured and analyzed. The gene signal intensities were normalized to tRNA signal intensity. Results: The primary MM cells and HMCLs displayed a distinctive miRNA expression profile compared with normal plasma cells. Using the GeneSpring 7 (Agilent Technologies, Palo Alto, CA) for gene expression analysis we have identified miRNA genes with significant variation in expression levels between tumor and normal samples including miR-125b, miR-133a, miR1, and miR-124a (p<0.01). Recently defined algorithms were used to identify putative targets for the miRNA genes of interest such as leukemia inhibitory factor (LIF) as targets of miR-125b and Stat3 and angiopoietin-1 precursor as targets of miR-124a (Lewis et al. 2003). Of note, miR-15 and miR-16, previously identified to be downregulated in CLL, were expressed at low levels in some MM patients and HMCLs, but not in the normal plasma cells. Further analysis will be carried out to validate the data from miRNA profiling using northern blot analysis or real-time RT-PCR to measure expression levels of miRNA genes and that of their target genes. Conclusion: Analysis of miRNA expression pattern (along with aCGH and gene expression profiling studies) will undoubtedly refine our understanding of the various genotypic subtypes of multiple myeloma.

Abstract BACKGROUND: Green tea has long been touted as a health promoting substance. The active chemical compounds in green tea are called polyphenols or catechins. Epigallocatechin gallate(EGCG) is the major catechin in green tea. We previously reported the in vitro ability of EGCG to induce apoptotic cell death in chronic lymphocytic leukemia(CLL) B-cells in vitro (Blood 104:788). After publication of our findings, clinical activity in individuals using over the counter green tea extracts were reported (Leuk Res 30:707). Based on this information, we opened a phase I/II trial of green tea extracts for patients with asymptomatic, early stage CLL in fall of 2005. METHODS: The purpose of the phase I portion of this trial was to determine the optimal dose of EGCG in the Polyphenon E preparation for chronic daily administration and define tolerability in CLL patients. Previously untreated patients with asymptomatic, Rai stage 0–II CLL not currently meeting National Cancer Institute(NCI) Working Group(WG) Criteria for treatment were eligible for participation. Polyphenon E with a standardized dose of EGCG was obtained from NCI. The phase I portion of the trial was designed with 8 dose levels(range 400–2000 mg orally BID) using the standard 3 patient per dose level design. Patients remained on study up to 6 months. Grade 2 adverse events attributed to study treatment that did not respond to supportive care were considered dose limiting toxicity. The trial was designed to administer Polyphenon E in the fasting state. After accrual to dose levels 1 and 2, the U.S. FDA mandated all U.S. trials of Polyphenon E administer drug in the fed state. Accordingly, drug was administered in the fed state for dose levels 3–8. Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the NCI WG Criteria. RESULTS: As of August 2007, 33 patients have been accrued to dose levels 1–8. The maximum tolerated dose(MTD) has not been reached. Side effects have generally been mild. The most common toxicities were nausea(grade 1: 42%; grade 2: 3%), elevation in SGOT (42%; all grade 1), and abdominal pain (36%; all grade 1). To date, no patient has had a sustained 50% reduction in both absolute lymphocyte count (ALC) and lymphadenopathy that would meet the NCI WG criteria for partial response. A majority of patients have had a reduction in ALC(Table). Among the 10 patients who had palpable adenopathy at study enrollment, 7 patients experienced at least a 50% reduction in the sum of the products of all nodal areas at some point during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.93974 ng/mL(median 40.5 ng/mL). Plasma levels did not clearly relate to the degree of reduction in ALC suggesting sensitivity to Polyphenon E may relate more to characteristics of the leukemic clone than plasma EGCG levels. CONCLUSION: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. The MTD has not been reached. As classified by the NCI WG criteria, no partial or complete remissions have been observed to date, however declines in ALC and lymphadenopathy have been observed in the majority of patients. The phase II portion of this trial will open at Mayo Clinic Fall 2007. Best reduction in ALC n % of patients At least 10% decline 25 76% At least 20% decline 14 42% At least 30% decline 8 24% At least 40% decline 4 12% At least 50% decline 2 6%

Abstract From a high throughput RNAi screen of the human druggable genome targeting the KMS11 cell line, we identified the suppression of the vacuolar H+- ATPase (V-ATPase) family as cytotoxic to myeloma cells. In the screen, two oligos against each gene for the V-ATPase subunits ATP6V1A and ATP6V1B1 resulted in suppression of cell growth (50% and 60% inhibition of cell viability respectively). We further confirmed this result using both lentiviral shRNA knockdown and two small molecule inhibitors specific for V-ATPase. Silencing of ATP6V1A by lentiviral shRNA knock-down in KMS11 and in OPM1 myeloma cell lines caused 75–80% reduction of cell viability at 5 days post infection (measured by MTT assay). Consistent with this result, the V-ATPase specific inhibitors, bafilomycin A1 and REATA 203, both inhibited the growth of a genetically heterogeneous and standardized panel of 14 human myeloma cell lines in vitro with an IC50 ranging from 2.2 – 8.9 nM (mean 5.25 nM) for bafilomycin A1 and 46–1594 nM (mean 542.5 nM)) for REATA 203. We further demonstrated that patient samples (n=10) were sensitive to 20nM bafilomycin A1 which induced a mean of 58% of MM cells to undergo apoptosis (range 10% to 93%) after 24 hours of treatment. Similar to bafilomycin A1, treatment of primary patient-derived MM cells with 500 nM REATA 203 for 72 hours resulted in a mean 69% apoptosis (range 24% to 97%). In contrast, non-myeloma cells (the CD138- fractions of the bone marrow samples) were less sensitive - mean 9% apoptosis (range from 0% to 34%) under the same treatment conditions. Of high interest, however, unlike most drugs we have studied in pre-clinical myeloma models, the cytotoxicity induced by bafilomycin A1 in MM cell lines is abrogated by co-culture with patient bone marrow stromal cells but is not affected by IL-6 or IGF-1 treatment. Dexamethasone- or melphalan-resistant MM cell lines were also highly sensitive to both bafilomycin A1 and REATA 203. In a xenographic JJN3 mouse model, bafilomycin A1 suppresses and delays growth of tumor in a dose-dependent fashion. Gene expression analysis of normal-donor bone marrow plasma cells (n=19), primary tumor samples from MM patients (n=107) and normal somatic tissues demonstrates ubiquitous expression of most subunits of V-ATPase, however, some subunits are preferentially expressed in myeloma cells compared with normal plasma cells, including ATP6V1F (84% vs. 11%), ATP6V1E1 (29% vs. 5%), ATP6V1G2 (17% vs. 0%) and ATP6V0E 2 (36% vs. 16%). In conclusion, our data indicate that vacuolar H+-ATPase inhibitors are of interest as potential therapeutics for MM.

Abstract Genetic abnormalities are important in the pathobiology of myeloma and established prognostic factors. High-density aCGH allow global unbiased detection of genomic gains and losses. In this study, we attempt to identify survival critical genomic aberrations by correlating aCGH-defined abnormalities with survival in 2 different cohorts of patients. aCGH was performed in 64 MM patients treated with high dose therapy from the Mayo Clinic using Agilent’s 44k DNA microarray genechip. In addition, a publicly available aCGH dataset performed in 67 MM patients treated with total therapy from the University of Arkansas Medical School using the lower density Agilent 22k chip (Human 1A v2) was also analyzed. Base 2 logarithms of the ratios between red and green intensities (log-ratios) were treated as a primary measure of copy number ratio between tumor and normal samples. To detect the regions of genomic aberration, values of a sliding window of 15 consecutive log-ratios were compared with those of an artificial ‘reference’ chromosome using the Wilcoxon rank sum test. The reference chromosome, constructed for each sample, was assembled by collecting log-ratio values close to zero. For this, 15 randomly sampled log-ratios from autosomal regions whose p-value of Wilcoxon signed rank test against 0 was larger than 0.05 were recursively accepted until the number of log-ratios in the assembled reference chromosome reached 1000. The p-values and their corresponding false discovery rates (q-values) for the copy number gain or loss were calculated independently for each sliding window and assigned to the center position of corresponding sliding window. The survival difference was examined between samples that had gained or lost a chromosomal region (sliding window) and samples that had not using the method of Kaplan-Meier and the statistical significance was assessed by the log-rank test. The sliding window was identified as gained or lost if P-value < 10 −4 and Q-value < 10[suo]-3. Sliding windows whose gains or losses led to poor survival with log-rank test p-value < 0.5, corresponding q-value < 0.5, and happened to ≥ 10% of all samples were retained. Several regions of genomic gains and losses were significantly associated with poorer survival with 5 deletion hotspots common between the 2 datasets (Table). We validated the prognostic significance of these survival critical loci using FISH in an independent dataset of 169 patients treated with high dose therapy. Clustering of these survival critical loci revealed clusters of MM characterized by low, intermediate or high number of these abnormalities, which are independent of ploidy and translocations subtypes. These subgroups have significantly different survival. In addition, the clustering analysis suggests the chromosome 13 loss is an early event whereas chromosome 1 abnormalities are late events. Our strategy therefore identified novel regions of genomic aberration that are important prognostically. However, it is likely that these regions are manifestation of genomically unstable tumors with complex abnormalities that have poorer prognosis. Chr Start End Length Cytoband 1 74,137,824 75,972,544 1,834,720 p31.1 1 62,433,123 84,792,809 22,359,686 p31.3 – p22.3 20 4,100,452 6,189,410 2,088,958 p13 – p12.3 20 6,717,170 13,142,205 6,425,035 p12.3 – p12.1 20 4,708,668 10,566,333 5,857,665 p13 – p12.2

ObjectiveObservational studies point to an inverse correlation between low‐density lipoprotein (LDL) cholesterol levels and risk of intracerebral hemorrhage (ICH), but it remains unclear whether this association is causal. We tested the hypothesis that genetically elevated LDL is associated with reduced risk of ICH.MethodsWe constructed one polygenic risk score (PRS) per lipid trait (total cholesterol, LDL, high‐density lipoprotein [HDL], and triglycerides) using independent genomewide significant single nucleotide polymorphisms (SNPs) for each trait. We used data from 316,428 individuals enrolled in the UK Biobank to estimate the effect of each PRS on its corresponding trait, and data from 1,286 ICH cases and 1,261 matched controls to estimate the effect of each PRS on ICH risk. We used these estimates to conduct Mendelian Randomization (MR) analyses.ResultsWe identified 410, 339, 393, and 317 lipid‐related SNPs for total cholesterol, LDL, HDL, and triglycerides, respectively. All four PRSs were strongly associated with their corresponding trait (all p < 1.00 × 10‐100). While one SD increase in the PRSs for total cholesterol (odds ratio [OR] = 0.92; 95% confidence interval [CI] = 0.85–0.99; p = 0.03) and LDL cholesterol (OR = 0.88; 95% CI = 0.81–0.95; p = 0.002) were inversely associated with ICH risk, no significant associations were found for HDL and triglycerides (both p > 0.05). MR analyses indicated that 1mmol/L (38.67mg/dL) increase of genetically instrumented total and LDL cholesterol were associated with 23% (OR = 0.77; 95% CI = 0.65–0.98; p = 0.03) and 41% lower risks of ICH (OR = 0.59; 95% CI = 0.42–0.82; p = 0.002), respectively.InterpretationGenetically elevated LDL levels were associated with lower risk of ICH, providing support for a potential causal role of LDL cholesterol in ICH. ANN NEUROL 2020 ANN NEUROL 2020;88:56–66

Coronavirus disease 2019 (COVID-19) is a global pandemic that is wreaking havoc on the health and economy of much of human civilization. Electrophysiologists have been impacted personally and professionally by this global catastrophe. In this joint article from representatives of the Heart Rhythm Society, the American College of Cardiology, and the American Heart Association, we identify the potential risks of exposure to patients, allied healthcare staff, industry representatives, and hospital administrators. We also describe the impact of COVID-19 on cardiac arrhythmias and methods of triage based on acuity and patient comorbidities. We provide guidance for managing invasive and noninvasive electrophysiology procedures, clinic visits, and cardiac device interrogations. In addition, we discuss resource conservation and the role of telemedicine in remote patient care along with management strategies for affected patients.

6016 Background: A germline mutation in let-7 complementary site 6 (LCS6) within the KRAS 3'-untranslated region (rs61764370, the KRAS-variant: TG/GG) is known to associate with poor outcome and drug resistance in various cancers compared to the wild type allele (TT). We examine the prognostic significance of the KRAS-variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Methods: The KRAS-variant was determined in 116 tumor DNA samples from HNSCC patients enrolled in 3 clinical trials and a tissue collection study using a previously validated PCR-based assay. Results: KRAS-variant status could be determined in 108/116 (93%) samples and an allele frequency of TG/GG was 28.7%. These results were correlated with patient demographics, p16/human papillomavirus (HPV) status and clinical outcome. There was no association between p16/HPV status and the KRAS-variant status (Fisher’s exact test, p=1.0). The KRAS-variant was associated with poor progression-free survival in patients treated with cisplatin+/-cetuximab (log-rank p=0.002) but this association was not observed in docetaxel/bortezomib treated patients (log-rank p=0.89). Conclusions: KRAS-variant is a potentially promising biomarker of poor prognosis and a predictive biomarker of cisplatin resistance in R/M HNSCC. Prospective validation is warranted. Clinical trial information: NCT00003809.