Citations

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients. To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients. We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Abstract Primary immunodeficiencies (PIDs) are a rare and heterogeneous group of inherited genetic disorders that are characterized by an absent or impaired immune system. In this report, we describe the use of next-generation sequencing to investigate a male infant with clinical and immunological manifestations suggestive of a PID. Whole-exome sequencing of the infant along with his parents revealed a novel nucleotide variant (cytosine to adenine substitution at nucleotide position 252) in the coding region of the interleukin 2 receptor subunit gamma ( IL2RG ) gene. The mother was found to be a carrier. These findings are consistent with a diagnosis of X-linked severe combined immunodeficiency and represent the first such reported mutation in an Indian family. This mutation leads to an asparagine to lysine substitution ( p.Asn84Lys ) located in the extracellular domain of IL2RG, which is predicted to be pathogenic. Our study demonstrates the power of next-generation sequencing in identifying potential causative mutations to enable accurate clinical diagnosis, prenatal screening, and carrier female detection in PID patients. We believe that this approach, which is not a current routine in clinical practice, will become a mainstream component of individualized medicine in the near future.

Objective To examine the association between maternal obesity on left ventricular (LV) size and recovery in women with peripartum cardiomyopathy (PPCM). Study Design This was a prospective analysis of 100 women enrolled within 13 weeks of PPCM diagnosis and followed for a year in the Investigation of Pregnancy Associated Cardiomyopathy study. Adiposity was defined by standard body mass index (BMI) definitions for under/normal weight, overweight, and obesity. Demographic, clinical, and biomarker variables were compared across weight categories. Outcomes LV end-diastolic diameter (LVEDD) and ejection fraction were measured at entry, 6, and 12 months postpartum. Multivariable regression models examined the relationship between adiposity, LV size, and leptin levels with cardiac recovery at 6 and 12 months postpartum. Results Obese and nonobese women had similar LV dysfunction at entry. Obese women had greater LV size and less LV recovery at 6 and 12 months postpartum. BMI was positively associated with leptin and ventricular diameter. Greater BMI at entry remained associated with less ventricular recovery at 6 months (p = 0.02) in adjusted race-stratified models. LVEDD at entry predicted lower ejection fraction at 6 months (p < 0.001) and similarly at 12 months. Conclusion Obese women with PPCM had greater cardiac remodeling, higher leptin levels, and diminished cardiac recovery.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,330 in 2016, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency. However, the indolent MM developed in Vk*MYC mice rarely progresses to a malignant stage, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre (Vk) or NrasLSL Q61R/+; IgG1-Cre (Q61R). To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly malignant MM. The MM developed in some mice were restricted in bone marrow and spleen and thus mimicked majority of MM patients, while MM developed in other mice was primarily located in lymph nodes and represented patients with extramedullary MM. Both types of MM cells displayed hyperproliferation and hyperactivation of AKT and ERK pathways. They could be cultured for several weeks with their original stromal cells and in the presence of Il-6. The cultured MM cells could be easily infected or transfected. Upon transplantation, both types of MM cells caused a rapid lethality (within 16 and 4 weeks respectively) in secondary and tertiary recipients and preserved the initial locations of donor MM cells. MM recipient mice developed bone lesions and/or fractures characteristic of MM patients. Preliminary preclinical studies indicated that both types of MM cells were resistant to Bortezomib, a proteasome inhibitor commonly used in treating MM patients, and AZD6244, a potent MEK inhibitor. Additional tests of combinatorial therapies are ongoing. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. This model can serve as a platform to investigate pathogenesis of highly malignant multiple myeloma and test the efficacy of novel therapeutic agents. Citation Format: Zhi Wen, Adhithi Rajagopalan, Erik A. Ranheim, Remington Finn, Adam Pagenkopf, Grant Yun, Yun Zhou, Yidan Wang, Demin Wang, Marta Chesi, P. Leif Bergsagel, Fotis Asimakopoulos, Jing Zhang. Expression of oncogenic Nras and a MYC transgene in germinal center B cells induces a highly malignant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3018.

Purpose: Optimal strategies to detect early interstitial lung disease (ILD) are unknown. ILD is frequently subpleural in distribution and affects lung elasticity. Lung ultrasound surface wave elastography (LUSWE) is a noninvasive method of quantifying superficial lung tissue elastic properties. In LUWSE a handheld device applied at the intercostal space vibrates the chest at a set frequency, and the lung surface wave velocity is measured by an ultrasound probe 5 mm away in the same intercostal space. We explored LUWSE’s ability to detect ILD and correlated LUSWE velocity with physiological, quantitative, and visual radiologic features of subjects with known ILD and of healthy controls. Materials and Methods: Seventy-seven subjects with ILD, mostly caused by connective tissue disease, and 19 healthy controls were recruited. LUSWE was performed on all subjects in 3 intercostal lung regions bilaterally. Comparison of LUSWE velocities pulmonary function testing, visual assessment, and quantitative analysis of recent computed tomographic imaging with Computer-Aided Lung Informatics for Pathology Evaluation and Rating (CALIPER) software. Results: Sonographic velocities were higher in all lung regions for cases, with the greatest difference in the lateral lower lung. Median velocity in m/s was 5.84 versus 4.11 and 5.96 versus 4.27 (P<0.00001) for cases versus controls, left and right lateral lower lung zones, respectively. LUSWE velocity correlated negatively with vital capacity and positively with radiologist and CALIPER-detected interstitial abnormalities. Conclusions: LUSWE is a safe and noninvasive technique that shows high sensitivity to detect ILD and correlated with clinical, physiological, radiologic, and quantitative assessments of ILD. Prospective study in detecting ILD is indicated.

Abstract Rationale Quantitative computed tomographic (CT) measures of baseline disease severity might identify patients with idiopathic pulmonary fibrosis (IPF) with an increased mortality risk. We evaluated whether quantitative CT variables could act as a cohort enrichment tool in future IPF drug trials. Objectives To determine whether computer-derived CT measures, specifically measures of pulmonary vessel–related structures (VRSs), can better predict functional decline and survival in IPF and reduce requisite sample sizes in drug trial populations. Methods Patients with IPF undergoing volumetric noncontrast CT imaging at the Royal Brompton Hospital, London, and St. Antonius Hospital, Utrecht, were examined to identify pulmonary function measures (including FVC) and visual and computer-derived (CALIPER [Computer-Aided Lung Informatics for Pathology Evaluation and Rating] software) CT features predictive of mortality and FVC decline. The discovery cohort comprised 247 consecutive patients, with validation of results conducted in a separate cohort of 284 patients, all fulfilling drug trial entry criteria. Measurements and Main Results In the discovery and validation cohorts, CALIPER-derived features, particularly VRS scores, were among the strongest predictors of survival and FVC decline. CALIPER results were accentuated in patients with less extensive disease, outperforming pulmonary function measures. When used as a cohort enrichment tool, a CALIPER VRS score greater than 4.4% of the lung was able to reduce the requisite sample size of an IPF drug trial by 26%. Conclusions Our study has validated a new quantitative CT measure in patients with IPF fulfilling drug trial entry criteria—the VRS score—that outperformed current gold standard measures of outcome. When used for cohort enrichment in an IPF drug trial setting, VRS threshold scores can reduce a required IPF drug trial population size by 25%, thereby limiting prohibitive trial costs. Importantly, VRS scores identify patients in whom antifibrotic medication prolongs life and reduces FVC decline.

Abstract TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.

ABSTRACT Background and objective This study evaluated whether patients with combined pulmonary fibrosis and emphysema (CPFE) have an increased likelihood of pulmonary hypertension (PHT) when compared with idiopathic pulmonary fibrosis (IPF) patients without emphysema. Methods Two consecutive IPF populations having undergone transthoracic echocardiography were examined ( n = 223 and n = 162). Emphysema and interstitial lung disease (ILD) extent were quantified visually; ILD extent was also quantified by a software tool, CALIPER. Echocardiographic criteria categorized PHT risk. Results The prevalence of an increased PHT likelihood was 29% and 31% in each CPFE cohort. Survival at 12 months was 60% across both CPFE cohorts with no significantly worsened outcome identified when compared with IPF patients without emphysema. Using logistic regression models in both cohorts, total computed tomography (CT) disease extent (ILD and emphysema) predicted the likelihood of PHT. After adjustment for total disease extent, CPFE had no stronger association with PHT likelihood than IPF patients without emphysema. Conclusion Our findings indicate that the reported association between CPFE and PHT is explained by the summed baseline CT extents of ILD and emphysema. Once baseline severity is taken into account, CPFE is not selectively associated with a malignant microvascular phenotype, when compared with IPF patients without emphysema.