ALBERT

Description: The cytokinesis-block micronucleus (CBMN) assay can be used to quantify micronucleus (MN) formation, the outcome measured being MN frequency. MN frequency has been shown to be both an accurate measure of chromosomal instability/DNA damage and a risk factor for cancer. Similarly, the Agilent 4 × 44k human oligonucleotide microarray can be used to quantify gene expression changes. Despite the existence of accepted methodologies to quantify both MN frequency and gene expression, very little is known about the association between the two. In modeling our count outcome (MN frequency) using gene expression levels from the high-throughput assay as our predictor variables, there are many more variables than observations. Hence, we extended the generalized monotone incremental forward stagewise method for predicting a count outcome for high-dimensional feature settings.

Description: Breast cancer (BC) is the second most common cancer among women. Research shows many women with BC experience anxiety, depression, and stress (ADS). Epigenetics has recently emerged as a potential mechanism for the development of depression.1 Although there are growing numbers of research studies indicating that epigenetic changes are associated with ADS, there is currently no evidence that this association is present in women with BC. The goal of this study was to identify high-throughput methylation sites (CpG sites) that are associated with three psychoneurological symptoms (ADS) in women with BC. Traditionally, univariate models have been used to examine the relationship between methylation sites and each psychoneurological symptom; nevertheless, ADS can be treated as a cluster of related symptoms and included together in a multivariate linear model. Hence, an overarching goal of this study is to compare and contrast univariate and multivariate models when identifying methylation sites associated with ADS in women with BC. When fitting separate linear regression models for each ADS scale, 3 among 285,173 CpG sites tested were significantly associated with depression. Two significant CpG sites are located on their respective genes FAM101A and FOXJ1, and the third site cannot be mapped to any known gene at this time. In contrast, the multivariate models identified 8,535 ADS-related CpG sites. In conclusion, when analyzing correlated psychoneurological symptom outcomes, multivariate models are more powerful and thus are recommended.

Description: The pathological description of the stage of a tumor is an important clinical designation and is considered, like many other forms of biomedical data, an ordinal outcome. Currently, statistical methods for predicting an ordinal outcome using clinical, demographic, and high-dimensional correlated features are lacking. In this paper, we propose a method that fits an ordinal response model to predict an ordinal outcome for high-dimensional covariate spaces. Our method penalizes some covariates (high-throughput genomic features) without penalizing others (such as demographic and/or clinical covariates). We demonstrate the application of our method to predict the stage of breast cancer. In our model, breast cancer subtype is a nonpenalized predictor, and CpG site methylation values from the Illumina Human Methylation 450K assay are penalized predictors. The method has been made available in the ordinalgmifs package in the R programming environment.

Description: High-throughput genomic assays are performed using tissue samples with the goal of classifying the samples as normal 〈 pre-malignant 〈 malignant or by stage of cancer using a small set of molecular features. In such cases, molecular features monotonically associated with the ordinal response may be important to disease development; that is, an increase in the phenotypic level (stage of cancer) may be mechanistically linked through a monotonic association with gene expression or methylation levels. Though traditional ordinal response modeling methods exist, they assume independence among the predictor variables and require the number of samples (n) to exceed the number of covariates (P) included in the model. In this paper, we describe our ordinalgmifs R package, available from the Comprehensive R Archive Network, which can fit a variety of ordinal response models when the number of predictors (P) exceeds the sample size (n). R code illustrating usage is also provided.

Description: No abstract supplied.

Description: Researchers have recently shown that penalized models perform well when applied to high-throughput genomic data. Previous researchers introduced the generalized monotone incremental forward stagewise (GMIFS) method for fitting overparameterized logistic regression models. The GMIFS method was subsequently extended by others for fitting several different logit link ordinal response models to high-throughput genomic data. In this study, we further extended the GMIFS method for ordinal response modeling using a complementary log-log link, which allows one to model discrete survival data. We applied our extension to a publicly available microarray gene expression dataset (GSE53733) with a discrete survival outcome. The dataset included 70 primary glioblastoma samples from patients of the German Glioma Network with long-, intermediate-, and short-term overall survival. We tested the performance of our method by examining the prediction accuracy of the fitted model. The method has been implemented as an addition to the ordinalgmifs package in the R programming environment.

Description: Key Points Anticoagulants inhibit release of angiogenic proteins from platelets.

Description: Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Description: Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

Description: Background We hypothesized lenalidomide (len) related diarrhea (LRD) may correlate with activation of an immune response against multiple myeloma (MM) based on colon biopsies that showed crypt apoptosis reminiscent of GVHD in 3 MM patients with severe LRD but without prior allogeneic stem cell transplantation or gastrointestinal disorder. To investigate whether survival may be influenced by the presence of LRD we performed a retrospective chart review. Methods Patients who developed symptomatic MM on or after 1/1/2005 as defined by the start of anti-MM therapy and had been on len for at least 6 consecutive months by 12/31/2010 were included in the analysis. Significant LRD was considered present if the treating physician attributed the diarrhea to len and recommended supportive therapy in at least two clinic notes.  The first time treatment for LRD was recommended was used as the date of onset of LRD. As possible confounding factors age at the start of len therapy, number of prior regimens, prior high dose chemotherapy with autologous stem cell transplant (ASCT), and use of antineoplastic agents other than corticosteroids at any time during the continuous len therapy were collected. The primary outcome was overall survival (OS), which was calculated from the start of len. Fisher’s exact test, Mann-Whitney test, and the logrank test were used to compare characteristics and duration of len between patients with and without LRD. Proportional hazards models were used to assess the impact of LRD on OS. The lag between the start of len and development of LRD was accounted for by treating LRD as a time-varying covariate in these models. Results 161 patients were identified, 47 (29%) had LRD, and 59 (37%) died during follow up. LRD and no LRD groups were balanced for gender, age, number of prior regimens, prior transplant, and use of antineoplastic agents other than corticosteroids with len, but len treatment duration differed; LRD patients had received a median of  43.4 consecutive months of len (range 5.8-82.9) compared to 14.6 (range 5.9-89) for patients without LRD (p=0.001). Onset of LRD occurred after a median of 17.7 months (range 0.3-75.4) of len therapy. In multivariable analyses, development of LRD (HR 0.46, 95% C.I. 0.21-1.00, p=0.05) was associated with improved OS as were the number of prior therapies (0-2, vs 〉2, HR 0.16, 95% C.I. 0.08-0.32, p