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Competing Interests: Michael H. Tomasson: consulting or advisory roles for Janssen. Shinsuke Iida: honoraria from Bristol Myers Squibb, Celgene, Janssen, Pfizer, Sanofi, and Takeda; consulting or advisory roles for Janssen, Sanofi, and Takeda; and research funding from AbbVie, Amgen, Bristol Myers Squibb, Caelum Biosciences, Celgene, Daiichi Sankyo, Janssen, Ono, Otsuka, Sanofi, and Takeda. Ruben Niesvizky: consulting or advisory roles for Bristol Myers Squibb, GSK, Janssen, Karyopharm Therapeutics, and Takeda and research funding from Bristol Myers Squibb, GSK, Janssen, Karyopharm Therapeutics, and Takeda. Mohamad Mohty: honoraria from Amgen, Celgene; consulting or advisory roles for Adaptive Biotechnologies, GSK, Jazz Pharmaceuticals, MaaT Pharma, Novartis, Sanofi, and Xenikos; personal fees from Amgen, Astellas, Bristol Myers Squibb, Celgene, Pfizer, Stemline‐Menarini and Takeda; and speakers bureau roles for Janssen, Jazz Pharmaceuticals, and Sanofi. Nizar J. Bahlis: honoraria from AbbVie, Amgen, Celgene, Genentech/Roche, GSK, Janssen, Karyopharm Therapeutics, Sanofi, and Takeda; consulting or advisory roles for Amgen, Celgene, Janssen, Karyopharm Therapeutics, Pfizer, Sanofi, and Takeda; personal fees from AbbVie, Amgen, Celgene, Genentech/Roche, GSK, Janssen, Karyopharm Therapeutics, Sanofi, and Takeda; and patents, royalties, and/or other intellectual property interests with Celgene and Janssen. Joaquin Martinez‐Lopez: consulting or advisory roles for Bristol Myers Squibb, Janssen, and Novartis; research funding from Astellas and Bristol Myers Squibb; and speakers bureau roles for Bristol Myers Squibb, Janssen‐Cilag, and Roche. Paula Rodriguez‐Otero: consulting or advisory roles for AbbVie, Bristol Myers Squibb, GSK, Janssen, Pfizer, and Sanofi; personal fees from AbbVie, Celgene, GSK, H3 Biomedicine, Janssen, Pfizer, and Sanofi; travel and accommodations expenses paid by Pfizer; and speakers bureau roles for Bristol Myers Squibb, GSK, Janssen, and Sanofi. Alexander M. Lesokhin: honoraria from iTeos Therapeutics, Janssen, Legend Biotech, Pfizer, Sanofi, and Trillium Therapeutics; consulting or advisory roles for Pfizer, Trillium Therapeutics, and Arcellx; personal fees from iTeos Therapeutics, Janssen, Legend Biotech, Pfizer, Sanofi, and Trillium Therapeutics; research funding from Bristol Myers Squibb, Genentech, Janssen, Pfizer, Sanofi, and Trillium Therapeutics; and patents, royalties and/or other intellectual property interests with Serametrix. Andrea Viqueira, Eric Leip, Umberto Conte, and Sharon T. Sullivan: employment and stock ownership at Pfizer. Guenther Koehne: has no conflicts of interest.

Multiple myeloma (MM) is a plasma cell malignancy expressing B cell maturation antigen (BCMA). Elranatamab, a bispecific antibody, engages BCMA on MM and CD3 on T cells. The MagnetisMM-1 trial evaluated its safety, pharmacokinetics and efficacy. Primary endpoints, including the incidence of dose-limiting toxicities as well as objective response rate (ORR) and duration of response (DOR), were met. Secondary efficacy endpoints included progression-free survival (PFS) and overall survival (OS). Eighty-eight patients with relapsed or refractory MM received elranatamab monotherapy, and 55 patients received elranatamab at efficacious doses. Patients had received a median of five prior regimens; 90.9% were triple-class refractory, 29.1% had high cytogenetic risk and 23.6% received prior BCMA-directed therapy. No dose-limiting toxicities were observed during dose escalation. Adverse events included cytopenias and cytokine release syndrome. Exposure was dose proportional. With a median follow-up of 12.0 months, the ORR was 63.6% and 38.2% of patients achieving complete response or better. For responders, the median DOR was 17.1 months. All 13 patients evaluable for minimal residual disease achieved negativity. Even after prior BCMA-directed therapy, 53.8% achieved response. For all 55 patients, median PFS was 11.8 months, and median OS was 21.2 months. Elranatamab achieved durable responses, manageable safety and promising survival for patients with MM. ClinicalTrials.gov Identifier: NCT03269136 ., (© 2023. The Author(s).)

Elranatamab is a humanized B-cell maturation antigen (BCMA)-CD3 bispecific antibody. In the ongoing phase 2 MagnetisMM-3 trial, patients with relapsed or refractory multiple myeloma received subcutaneous elranatamab once weekly after two step-up priming doses. After six cycles, persistent responders switched to biweekly dosing. Results from cohort A, which enrolled patients without prior BCMA-directed therapy (n = 123) are reported. The primary endpoint of confirmed objective response rate (ORR) by blinded independent central review was met with an ORR of 61.0% (75/123); 35.0% ≥complete response. Fifty responders switched to biweekly dosing, and 40 (80.0%) improved or maintained their response for ≥6 months. With a median follow-up of 14.7 months, median duration of response, progression-free survival and overall survival (secondary endpoints) have not been reached. Fifteen-month rates were 71.5%, 50.9% and 56.7%, respectively. Common adverse events (any grade; grade 3-4) included infections (69.9%, 39.8%), cytokine release syndrome (57.7%, 0%), anemia (48.8%, 37.4%), and neutropenia (48.8%, 48.8%). With biweekly dosing, grade 3-4 adverse events decreased from 58.6% to 46.6%. Elranatamab induced deep and durable responses with a manageable safety profile. Switching to biweekly dosing may improve long-term safety without compromising efficacy. ClinicalTrials.gov identifier: NCT04649359 ., (© 2023. The Author(s).)

Venetoclax is a highly selective B-cell lymphoma-2 (BCL-2) inhibitor, which, combined with a DNA hypomethylating agent or low dose cytarabine, results in high rates of initial responses in patients with acute myeloid leukemia (AML). However, the disease relapses in most patients. Mechanisms of resistance to venetoclax-based therapy include TP53 gene mutations or inactivation of p53 protein, activating kinase mutations such as FLT3 and RAS, and upregulation of other BCL-2 family apoptotic proteins. Current clinical trials are exploring strategies such as doublet or triplet regimens incorporating a p53 activator, an anti-CD47 antibody, or other novel agents that target genes and proteins responsible for resistance to venetoclax. Further studies should focus on identifying predictive biomarkers of response to venetoclax-based therapy and incorporating immunotherapeutic approaches such as checkpoint inhibitors, bispecific antibodies, antibody-drug conjugates, and CAR T-cell therapy to improve outcomes for patients with AML., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chamber exposures are commonly used to evaluate the physiological and pathophysiological consequences of intermittent hypoxia in animal models. Researchers in this field use both commercial and custom-built chambers in their experiments. The purpose of this Cores of Reproducibility in Physiology paper is to demonstrate potential sources of variability in these systems that researchers should consider. Evaluating the relationship between arterial oxygen saturation and inspired oxygen concentration, we found that there are important sex-dependent differences in the commonly used C57BL6/J mouse model. The time delay of the oxygen sensor that provides feedback to the system during the ramp-down and ramp-up phases was different, limiting the number of cycles per hour that can be conducted and the overall stability of the oxygen concentration. The time to reach the hypoxic and normoxic hold stages, and the overall oxygen concentration, were impacted by the cycle number. These variables were further impacted by whether there are animals present in the chamber, highlighting the importance of verifying the cycling frequency with animals in the chamber. At ≤14 cycles/h, instability in the chamber oxygen concentration did not impact arterial oxygen saturation but may be important at higher cycle numbers. Taken together, these data demonstrate the important sources of variability that justify reporting and verifying the target oxygen concentration, cycling frequency, and arterial oxygen concentration, particularly when comparing different animal models and chamber configurations. NEW & NOTEWORTHY Intermittent hypoxia exposures are commonly used in physiology and many investigators use chamber systems to perform these studies. Because of the variety of chamber systems and protocols used, it is important to understand the sources of variability in intermittent hypoxia experiments that can impact reproducibility. We demonstrate sources of variability that come from the animal model, the intermittent hypoxia protocol, and the chamber system that can impact reproducibility.

This meta-analysis, which consisted of a scoping review and retrospective medical record review, is focused on potential sex differences in cardiovascular diseases in patients with Down syndrome. We limited our review to peer-reviewed, primary articles in the English language, in the PubMed and Web of Science databases from 1965 to 2021. Guidelines for scoping reviews were followed throughout the process. Four categorical domains were identified and searched using additional keywords: 1 ) congenital heart disease, 2 ) baseline physiology and risk factors, 3 ) heart disease and hypertension, and 4 ) stroke and cerebrovascular disease. Articles were included if they reported male and female distinct data, participants with Down syndrome, and one of our keywords. The retrospective medical record review was completed using 75 participating health care organizations to identify the incidence of congenital and cardiovascular diseases and to quantify cardiovascular risk factors in male and female patients. Female patients with Down syndrome are at higher risk of hypertension, ischemic heart disease, and cerebrovascular disease. The risk of congenital heart disease is higher in males with Down syndrome at all ages included in our analyses. Some of the male-to-female sex differences in cardiovascular disease risk in the general patient population are not present, or reversed in the Down syndrome population. This information should be considered for future investigations and ongoing patient care. NEW & NOTEWORTHY In patients with Down syndrome (DS), CHD is the leading cause of death <20 yr old and cardiovascular disease is a leading cause of death in individuals >20 yr old. Men with DS live longer than women. It is unknown if sex differences are present in cardiovascular disease and dysregulation in DS across the lifespan. We observed higher risk of hypertension, ischemic heart disease, and cerebrovascular disease in females and a higher risk of CHD in males with DS.

Diagnosing the cause of hypoxemia and dyspnea can be complicated in complex patients with multiple comorbidities. This "Case Study in Physiology" describes an man with obesity admitted to the hospital for relapse of acute lymphoblastic leukemia, who experienced progressive hypoxemia, shortness of breath, and dyspnea on exertion during his hospitalization. After initial empirical treatment with diuresis and antibiotics failed to improve his symptoms and because an arterial blood gas measurement was not readily available, we applied a novel, recently described physiological method to estimate the arterial partial pressure of oxygen from the peripheral saturation measurement and calculate the alveolar-arterial oxygen difference to discern the source of his hypoxemia and dyspnea. Using basic physiological principles, we describe how hypoventilation, anemia, and the use of a β blocker and furosemide, collaborated to create a "perfect storm" in this patient that impaired oxygen delivery and limited utilization. This case illustrates the application of innovative physiology methodology in medicine and provides a strong rationale for continuing to integrate physiology education in medical education. NEW & NOTEWORTHY Discerning the cause of dyspnea and hypoxemia in complex patients can be difficult. We describe the "real world" application of an innovative methodology to untangle the underlying physiology in a patient with multiple comorbidities. This case further demonstrates the importance of applying physiology to interrogate the underlying cause of a patient's symptoms when treatment based on probability fails.

Hematopoietic stem cell transplant (HSCT) is a potentially curative treatment for hematopoietic malignancies, complicated by decreased performance status and quality of life. Exercise therapy improves outcomes in HSCT, but several barriers have prevented exercise from becoming routine clinical practice. Based on existing data that wearable technologies facilitate exercise participation in other sedentary and chronic illness populations, we propose the novel hypothesis that wearable technologies are a valuable tool in transcending barriers and developing exercise therapy programs for HSCT patients., (Copyright © 2021 by the American College of Sports Medicine.)

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub-nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as silver-binding nucleolar organizing regions (AgNORs). Supporting these data, samples from t(4;14)-positive patients had higher AgNORs scores than t(4;14)-negative samples. ACA11 also upregulated ribosome production, pre-47S rRNA synthesis, and protein synthesis in a ROS-dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)-positive myeloma patients., Competing Interests: CONFLICT OF INTEREST The authors declare no competing financial interests.

Obesity is the only known modifiable risk factor for multiple myeloma (MM), an incurable cancer of bone marrow plasma cells. The mechanism linking the two is unknown. Obesity is associated with an increased risk of sleep apnea, which results in chronic intermittent hypoxia (CIH), and drives solid tumor aggressiveness. Given the link between CIH and solid tumor progression, we tested the hypothesis that CIH drives the proliferation of MM cells in culture and their engraftment and progression in vivo. Malignant mouse 5TGM1 cells were cultured in CIH, static hypoxia, or normoxia as a control in custom, gas-permeable plates. Typically MM-resistant C57BL/6J mice were exposed to 10 h/day CIH (AHI = 12/h), static hypoxia, or normoxia for 7 days, followed by injection with 5TGM1 cells and an additional 28 days of exposure. CIH and static hypoxia slowed the growth of 5TGM1 cells in culture. CIH-exposed mice developed significantly more MM than controls (67 vs. 12%, P = 0.005), evidenced by hindlimb paralysis, gammopathy, bone lesions, and bone tumor formation. Static hypoxia was not a significant driver of MM progression and did not reduce survival ( P = 0.117). Interestingly, 5TGM1 cells preferentially engrafted in the bone marrow and promoted terminal disease in CIH mice, despite a lower tumor burden, compared with the positive controls. These first experiments in the context of hematological cancer demonstrate that CIH promotes MM through mechanisms distinct from solid tumors and that sleep apnea may be a targetable risk factor in patients with or at risk for blood cancer.

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.

Purpose: Recent advances have uncovered therapeutic interventions that might reduce the risk of progression of premalignant diagnoses, such as monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM). It remains unclear how to best screen populations at risk and how to evaluate the ability of these interventions to reduce disease prevalence and mortality at the population level. To address these questions, we developed a computational modeling framework., Materials and Methods: We used individual-based computational modeling of MGUS incidence and progression across a population of diverse individuals to determine best screening strategies in terms of screening start, intervals, and risk-group specificity. Inputs were life tables, MGUS incidence, and baseline MM survival. We measured MM-specific mortality and MM prevalence after MGUS detection from simulations and mathematic modeling predictions., Results: Our framework is applicable to a wide spectrum of screening and intervention scenarios, including variation of the baseline MGUS to MM progression rate and evolving MGUS, in which progression increases over time. Given the currently available point estimate of progression risk reduction to 61% risk, starting screening at age 55 years and performing follow-up screening every 6 years reduced total MM prevalence by 19%. The same reduction could be achieved with starting screening at age 65 years and performing follow-up screening every 2 years. A 40% progression risk reduction per patient with MGUS per year would reduce MM-specific mortality by 40%. Specifically, screening onset age and screening frequency can change disease prevalence, and progression risk reduction changes both prevalence and disease-specific mortality. Screening would generally be favorable in high-risk individuals., Conclusion: Screening efforts should focus on specifically identified groups with high lifetime risk of MGUS, for which screening benefits can be significant. Screening low-risk individuals with MGUS would require improved preventions.

Multiple myeloma is an invariably fatal cancer of plasma cells. Despite tremendous advances in treatment, this malignancy remains incurable in most individuals. We postulate that strategies aimed at prevention have the potential to be more effective in preventing myeloma-related death than additional pharmaceutical strategies aimed at treating advanced disease. Here, we present a rationale for the development of prevention therapy and highlight potential target areas of study.

Multiple myeloma (MM) is a disease of copy number variants (CNVs), chromosomal translocations, and single-nucleotide variants (SNVs). To enable integrative studies across these diverse mutation types, we developed a capture-based sequencing platform to detect their occurrence in 465 genes altered in MM and used it to sequence 95 primary tumor-normal pairs to a mean depth of 104×. We detected cases of hyperdiploidy (23%), deletions of 1p (8%), 6q (21%), 8p (17%), 14q (16%), 16q (22%), and 17p (4%), and amplification of 1q (19%). We also detected IGH and MYC translocations near expected frequencies and non-silent SNVs in NRAS (24%), KRAS (21%), FAM46C (17%), TP53 (9%), DIS3 (9%), and BRAF (3%). We discovered frequent mutations in IGLL5 (18%) that were mutually exclusive of RAS mutations and associated with increased risk of disease progression (p = 0.03), suggesting that IGLL5 may be a stratifying biomarker. We identified novel IGLL5/IGH translocations in two samples. We subjected 15 of the pairs to ultra-deep sequencing (1259×) and found that although depth correlated with number of mutations detected (p = 0.001), depth past ~300× added little. The platform provides cost-effective genomic analysis for research and may be useful in individualizing treatment decisions in clinical settings.

Overexpression of the multiple myeloma set domain (MMSET) Wolf-Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t(4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138 + tumor cells from t(4;14)-positive and -negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t(4;14)-negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11-driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid-derived 2)-like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11-driven increases in ROS; however, transcriptional up-regulation of some of NRF2's antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.-Mahajan, N., Wu, H.-J., Bennett, R. L., Troche, C., Licht, J. D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA., (© FASEB.)

Background: Genome-wide association studies (GWAS) in European populations have identified genetic risk variants associated with multiple myeloma., Methods: We performed association testing of common variation in eight regions in 1,318 patients with multiple myeloma and 1,480 controls of European ancestry and 1,305 patients with multiple myeloma and 7,078 controls of African ancestry and conducted a meta-analysis to localize the signals, with epigenetic annotation used to predict functionality., Results: We found that variants in 7p15.3, 17p11.2, 22q13.1 were statistically significantly (P < 0.05) associated with multiple myeloma risk in persons of African ancestry and persons of European ancestry, and the variant in 3p22.1 was associated in European ancestry only. In a combined African ancestry-European ancestry meta-analysis, variation in five regions (2p23.3, 3p22.1, 7p15.3, 17p11.2, 22q13.1) was statistically significantly associated with multiple myeloma risk. In 3p22.1, the correlated variants clustered within the gene body of ULK4 Correlated variants in 7p15.3 clustered around an enhancer at the 3' end of the CDCA7L transcription termination site. A missense variant at 17p11.2 (rs34562254, Pro251Leu, OR, 1.32; P = 2.93 × 10 -7 ) in TNFRSF13B encodes a lymphocyte-specific protein in the TNF receptor family that interacts with the NF-κB pathway. SNPs correlated with the index signal in 22q13.1 cluster around the promoter and enhancer regions of CBX7 CONCLUSIONS: We found that reported multiple myeloma susceptibility regions contain risk variants important across populations, supporting the use of multiple racial/ethnic groups with different underlying genetic architecture to enhance the localization and identification of putatively functional alleles., Impact: A subset of reported risk loci for multiple myeloma has consistent effects across populations and is likely to be functional. Cancer Epidemiol Biomarkers Prev; 25(12); 1609-18. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: The molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear., Methods: We enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols., Results: Of the 116 patients, 53 (46%) had bone marrow blast clearance (<5% blasts). Response rates were higher among patients with an unfavorable-risk cytogenetic profile than among patients with an intermediate-risk or favorable-risk cytogenetic profile (29 of 43 patients [67%] vs. 24 of 71 patients [34%], P<0.001) and among patients with TP53 mutations than among patients with wild-type TP53 (21 of 21 [100%] vs. 32 of 78 [41%], P<0.001). Previous studies have consistently shown that patients with an unfavorable-risk cytogenetic profile and TP53 mutations who receive conventional chemotherapy have poor outcomes. However, in this study of 10-day courses of decitabine, neither of these risk factors was associated with a lower rate of overall survival than the rate of survival among study patients with intermediate-risk cytogenetic profiles., Conclusions: Patients with AML and MDS who had cytogenetic abnormalities associated with unfavorable risk, TP53 mutations, or both had favorable clinical responses and robust (but incomplete) mutation clearance after receiving serial 10-day courses of decitabine. Although these responses were not durable, they resulted in rates of overall survival that were similar to those among patients with AML who had an intermediate-risk cytogenetic profile and who also received serial 10-day courses of decitabine. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT01687400 .).

Purpose: Since the pioneering work of Salmon and Durie, quantitative measures of tumor burden in multiple myeloma have been used to make clinical predictions and model tumor growth. However, such quantitative analyses have not yet been performed on large datasets from trials using modern chemotherapy regimens., Experimental Design: We analyzed a large set of tumor response data from three randomized controlled trials of bortezomib-based chemotherapy regimens (total sample size n = 1,469 patients) to establish and validate a novel mathematical model of multiple myeloma cell dynamics., Results: Treatment dynamics in newly diagnosed patients were most consistent with a model postulating two tumor cell subpopulations, "progenitor cells" and "differentiated cells." Differential treatment responses were observed with significant tumoricidal effects on differentiated cells and less clear effects on progenitor cells. We validated this model using a second trial of newly diagnosed patients and a third trial of refractory patients. When applying our model to data of relapsed patients, we found that a hybrid model incorporating both a differentiation hierarchy and clonal evolution best explains the response patterns., Conclusions: The clinical data, together with mathematical modeling, suggest that bortezomib-based therapy exerts a selection pressure on myeloma cells that can shape the disease phenotype, thereby generating further inter-patient variability. This model may be a useful tool for improving our understanding of disease biology and the response to chemotherapy regimens. Clin Cancer Res; 22(16); 4206-14. ©2016 AACR., (©2016 American Association for Cancer Research.)

Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3'-5' exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, we find that absence of dis3 activity inhibits cell division. We uncover a conserved CDK1 phosphorylation site that when phosphorylated inhibits Dis3's exonuclease, but not endonuclease, activity. Leveraging this information, we show that Dis3's exonuclease function is required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, we find that modest reduction of dis3 function enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, we find that dis3 and ras genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms., (Copyright © 2016 by the Genetics Society of America.)

Unlabelled: Very-late-antigen-4 (VLA-4, α4β1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker., Methods: Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5., Results: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe., Conclusion: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

The retinoblastoma gene (RB1) has been implicated as a tumor suppressor in multiple myeloma (MM), yet its role remains unclear because in the majority of cases with 13q14 deletions, un-mutated RB1 remains expressed from the retained allele. To explore the role of Rb1 in MM, we examined the functional consequences of single- and double-copy Rb1 loss in germinal center B cells, the cells of origin of MM. We generated mice without Rb1 function in germinal center B cells by crossing Rb1(Flox/Flox) with C-γ-1-Cre (Cγ1) mice expressing the Cre recombinase in class-switched B cells in a p107(-/-) background to prevent p107 from compensating for Rb1 loss (Cγ1-Rb1(F/F)-p107(-/-)). All mice developed normally, but B cells with two copies of Rb1 deleted (Cγ1-Rb1(F/F)-p107(-/-)) exhibited increased proliferation and cell death compared with Cγ1-Rb1(+/+)-p107(-/-) controls ex vivo. In vivo, Cγ1-Rb1(F/F)-p107(-/-) mice had a lower percentage of splenic B220+ cells and reduced numbers of bone marrow antigen-specific secreting cells compared with control mice. Our data indicate that Rb1 loss induces both cell proliferation and death in germinal center B cells. Because no B-cell malignancies developed after 1 year of observation, our data also suggest that Rb1 loss is not sufficient to transform post-germinal center B cells and that additional, specific mutations are likely required to cooperate with Rb1 loss to induce malignant transformation., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Sn2 lipase labile phospholipid prodrugs in conjunction with contact-facilitated drug delivery offer an important advancement in Nanomedicine. Many drugs incorporated into nanosystems, targeted or not, are substantially lost during circulation to the target. However, favorably altering the pharmacokinetics and volume of distribution of systemic drug delivery can offer greater efficacy with lower toxicity, leading to new prolonged-release nanoexcipients. However, the concept of achieving Paul Erhlich's inspired vision of a 'magic bullet' to treat disease has been largely unrealized due to unstable nanomedicines, nanosystems achieving low drug delivery to target cells, poor intracellular bioavailability of endocytosed nanoparticle payloads, and the substantial biological barriers of extravascular particle penetration into pathological sites. As shown here, Sn2 phospholipid prodrugs in conjunction with contact-facilitated drug delivery prevent premature drug diffusional loss during circulation and increase target cell bioavailability. The Sn2 phospholipid prodrug approach applies equally well for vascular constrained lipid-encapsulated particles and micelles the size of proteins that penetrate through naturally fenestrated endothelium in the bone marrow or thin-walled venules of an inflamed microcirculation. At one time Nanomedicine was considered a 'Grail Quest' by its loyal opposition and even many in the field adsorbing the pains of a long-learning curve about human biology and particles. However, Nanomedicine with innovations like Sn2 phospholipid prodrugs has finally made 'made the turn' toward meaningful translational success., (© 2015 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.)

Costa et al. recently reported that racial disparities prevented nearly 40% of non-Hispanic blacks with multiple myeloma (MM) from undergoing stem cell transplantation (SCT), but the authors were unable to provide an explanation for the disparities because of limitations of their datasets. They hypothesized that socioeconomic status (SES) and/or insurance providers might account for the disparity. To examine the issue raised by Costa et al., we performed a secondary analysis using hierarchical multivariate logistic regression with data previously collected to determine if age at diagnosis, sex, SES, primary insurance provider at diagnosis, and comorbidity score help explain the racial disparities in SCT utilization. A model of race, age, sex, SES, insurance provider, and comorbidity score was the most accurate model in predicting stem cell utilization status (χ(2)[12] = 193.859; P < .001; area under the curve = .837; P < .001). After controlling for the covariates, black patients were less likely to undergo SCT than white patients (adjusted odds ratio, .49; 95% confidence interval, .27 to .89; P = .013). In conclusion, we also observed racial disparities between black and white patients with MM in SCT utilization and these are not fully accounted for by the covariates age, sex, SES, insurance provider, and comorbidity score., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Multiple myeloma pathogenesis is driven by the MYC oncoprotein, its dimerization with MAX, and the binding of this heterodimer to E-Boxes in the vicinity of target genes. The systemic utility of potent small molecule inhibitors of MYC-MAX dimerization was limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. We hypothesized that new lipid-based MYC-MAX dimerization inhibitor prodrugs delivered via integrin-targeted nanoparticles (NP) would overcome prior shortcomings of MYC inhibitor approaches and prolong survival in a mouse model of cancer. An Sn 2 lipase-labile prodrug inhibitor of MYC-MAX dimerization (MI1-PD) was developed which decreased cell proliferation and induced apoptosis in cultured multiple myeloma cell lines alone (P < 0.05) and when incorporated into integrin-targeted lipid-encapsulated NPs (P < 0.05). Binding and efficacy of NPs closely correlated with integrin expression of the target multiple myeloma cells. Using a KaLwRij metastatic multiple myeloma mouse model, VLA-4-targeted NPs (20 nm and 200 nm) incorporating MI1-PD (D) NPs conferred significant survival benefits compared with respective NP controls, targeted (T) no-drug (ND), and untargeted (NT) control NPs (T/D 200: 46 days vs., Nt/nd: 28 days, P < 0.05 and T/D 20: 52 days vs., Nt/nd: 29 days, P = 0.001). The smaller particles performed better of the two sizes. Neither MI1 nor MI1-PD provided survival benefit when administered systemically as free compounds. These results demonstrate for the first time that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using a targeted nanotherapy approach., (©2015 American Association for Cancer Research.)

Monoclonal gammopathy of undetermined significance (MGUS) is the requisite precursor to multiple myeloma (MM), a malignancy of antibody-producing plasma B-cells. The genetic basis of MGUS and its progression to MM remains poorly understood. C57BL/KaLwRij (KaLwRij) is a spontaneously-derived inbred mouse strain with a high frequency of benign idiopathic paraproteinemia (BIP), a phenotype with similarities to MGUS including progression to MM. Using mouse haplotype analysis, human MM SNP array data, and whole exome and whole genome sequencing of KaLwRij mice, we identified novel KaLwRij gene variants, including deletion of Samsn1 and deleterious point mutations in Tnfrsf22 and Tnfrsf23. These variants significantly affected multiple cell types implicated in MM pathogenesis including B-cells, macrophages, and bone marrow stromal cells. These data demonstrate that multiple cell types contribute to MM development prior to the acquisition of somatic driver mutations in KaLwRij mice, and suggest that MM may an inherently non-cell autonomous malignancy.

Aims: The activity of the transcription factor c-Myc is dependent upon heterodimerization with Max to control target gene transcription. Small-molecule inhibitors of c-Myc-Max have exhibited low potency and poor water solubility and are therefore unsuitable for in vivo application. We hypothesized that a nanomedicine approach incorporating a cryptic c-Myc inhibitor prodrug could be delivered and enzymatically released in order to effectively inhibit melanoma., Materials & Methods: An Sn-2 lipase-labile Myc inhibitor prodrug was synthesized and included in two αvβ3-targeted nanoparticle platforms (20 and 200 nm). The inherent antiproliferate potency was compared with the lipid-free compound using human and mouse melanoma cell lines., Results & Conclusion: These data demonstrate for the first time a successful nanodelivery of c-Myc inhibitors and their potential use to prevent melanoma.

Population-based studies suggest that black patients with multiple myeloma (MM) have a higher mortality rate than white patients. However, other studies suggest that this disparity is related to socioeconomic status (SES) rather than race. To provide clarity on this topic, we reviewed 562 patients diagnosed with MM at our institution. Patients with high SES had a median overall survival (OS) of 62.8 months (95% confidence interval [CI] 43.1-82.6 months), compared to 53.7 months (45.2-62.3 months) and 48.6 months (40.4-56.8 months) for middle and low SES, respectively (p = 0.015). After controlling for race, age, year of diagnosis, severity of comorbidities, stem cell transplant utilization and insurance provider, patients with low SES had a 54% increase in mortality rate relative to patients with high SES. To support our findings, we performed a similar analysis of 45,505 patients with MM from the Surveillance, Epidemiology and End Results-18 (SEER) database. Low SES is independently associated with poorer OS in MM.

The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.

Despite tremendous advances in treatments for myeloma in the past decade, the disease remains incurable in the majority of patients. Here, we review recent data demonstrating an association between obesity and increased risk of myeloma development. This may be due to the pro-inflammatory cytokine profile caused by obesity. Currently, there are no screening or prevention strategies for myeloma, but we propose that obesity-associated inflammatory pathways, or obesity itself, may be amenable to intervention, thereby preventing the transition from pre-malignancy to myeloma. In addition, we suggest that the morbidity, mortality and the significant costs associated with myeloma treatment could be reduced by addressing modifiable risk factors, and that research efforts should explore this novel hypothesis.

Patients with multiple myeloma who are refractory or intolerant to both bortezomib and lenalidomide have a poor prognosis. Next-generation therapies carfilzomib and pomalidomide have shown promising activity in this dual refractory population. Here we describe the clinical characteristics and ascertain the effects of carfilzomib and pomalidomide on survival in this patient cohort. We retrospectively reviewed the records of 65 patients with dual refractory/intolerant myeloma diagnosed between January 2007 and May 2012 at a single institution. The median overall survival (OS) from the time patients became dual refractory/intolerant was 10.2 months. Patients who received carfilzomib or pomalidomide after they became dual refractory/intolerant had a better OS compared to those who did not (12.6 vs. 6.8 months, p = 0.03 by Wilcoxon test). Prospective randomized control trials are needed for confirmation.

Activating mutations in Ras (N- and K-) are the most common point mutations found in patients with multiple myeloma (MM) and are associated with poor clinical outcome. We sought to directly examine the role of Ras activation in MM pathogenesis and used two different tissue-specific Cre recombinase mouse lines (Cγ1-Cre and AID-Cre), to generate mice with mutant Kras (Kras(G12D) ) activated specifically in germinal center B-cells. We also generated mice with activation of the Kras(G12D) allele in a tumor-prone Arf-null genetic background. Surprisingly, we observed no significant disruption in B-cell homeostasis in any of these models by serum immunoglobulin ELISA, SPEP, flow cytometry and histological examination. We observed development of non-overlapping tumor types due to off-target Cre expression, but despite successful recombination in germinal center and later B-cell populations, we observed no B-cell phenotype. Together, these data demonstrate that Ras activation is not sufficient to transform primary germinal center B-cells, even in an Arf-null context, and that the temporal order of mutation acquisition may be critical for myeloma development. Specific pathways, yet to be identified, are required before Kras can contribute to the development of MM.

Background: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear., Methods: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis., Results: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories., Conclusions: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.

Biomedical imaging techniques such as skeletal survey and (18)F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30-50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α(4)β(1) integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, (64)Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of (64)Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with (64)Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, (64)Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using (64)Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.

Purpose: We investigated the association between body mass index (BMI) at the time of multiple myeloma (MM) diagnosis and overall survival in a cohort of patients within the Veterans Health Administration system. We also evaluated the association between weight loss in the year prior to diagnosis and survival., Patients and Methods: Prospective analysis was performed on a retrospectively assembled cohort of 2,968 U.S. veterans diagnosed and treated for MM between September 1, 1999, and September 30, 2009, with follow-up information through October 22, 2011. Cox modeling controlling for patient- and disease-related prognostic variables was used to analyze the data., Results: Underweight patients (BMI <18.5 kg/m2) had increased mortality, whereas patients who were overweight (BMI 25-29.9 kg/m2) and obese (BMI ≥30 kg/m2) had lower mortality compared with healthy-weight patients (BMI 18.5-24.9 kg/m2). Weight loss ≥10% of baseline in the year before diagnosis was also associated with increased mortality and made the association between increased BMI and survival nonsignificant., Conclusion: Disease-related weight loss may be an important and heretofore unknown indicator of poor prognosis in MM. Assessment of weight loss prior to MM diagnosis should become a standard component of the clinical history in patients with newly diagnosed MM. Further research may identify relationships between disease-related weight loss and currently used prognostic factors in MM, further defining the role of this clinical factor in prognostic stratification.

Multiple myeloma (MM) is an incurable, B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in bone marrow. Despite recent advances in the understanding of genomic aberrations, a comprehensive catalogue of clinically actionable mutations in MM is just beginning to emerge. The tyrosine kinase (TK) and RAS oncogenes, which encode important regulators of various signaling pathways, are among the most frequently altered gene families in cancer. To clarify the role of TK and RAS genes in the pathogenesis of MM, we performed a systematic, targeted screening of mutations on prioritized RAS and TK genes, in CD138-sorted bone marrow specimens from 42 untreated patients. We identified a total of 24 mutations in the KRAS, PIK3CA, INSR, LTK, and MERTK genes. In particular, seven novel mutations in addition to known KRAS mutations were observed. Prediction analysis tools PolyPhen and Sorting Intolerant from Tolerant (SIFT) were used to assess the functional significance of these novel mutations. Our analysis predicted that these mutations may have a deleterious effect, resulting in the functional alteration of proteins involved in the pathogenesis of myeloma. While further investigation is needed to determine the functional consequences of these proteins, mutational testing of the RAS and TK genes in larger myeloma cohorts might also be useful to establish the recurrent nature of these mutations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The histone methyltransferase WHSC1 (also known as MMSET) is overexpressed in multiple myeloma (MM) as a result of the t(4;14) chromosomal translocation and in a broad variety of other cancers by unclear mechanisms. Overexpression of WHSC1 did not transform wild-type or tumor-prone primary hematopoietic cells. We found that ACA11, an orphan box H/ACA class small nucleolar RNA (snoRNA) encoded within an intron of WHSC1, was highly expressed in t(4;14)-positive MM and other cancers. ACA11 localized to nucleoli and bound what we believe to be a novel small nuclear ribonucleoprotein (snRNP) complex composed of several proteins involved in postsplicing intron complexes. RNA targets of this uncharacterized snRNP included snoRNA intermediates hosted within ribosomal protein (RP) genes, and an RP gene signature was strongly associated with t(4;14) in patients with MM. Expression of ACA11 was sufficient to downregulate RP genes and other snoRNAs implicated in the control of oxidative stress. ACA11 suppressed oxidative stress, afforded resistance to chemotherapy, and increased the proliferation of MM cells, demonstrating that ACA11 is a critical target of the t(4;14) translocation in MM and suggesting an oncogenic role in other cancers as well.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.

Context: Whole-genome sequencing is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis., Objective: To determine whether whole-genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame., Design, Setting, and Patient: We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase fluorescence in situ hybridization (FISH). The case patient was enrolled in an institutional review board-approved protocol, with consent specifically tailored to the implications of whole-genome sequencing. The protocol uses a "movable firewall" that maintains patient anonymity within the entire research team but allows the research team to communicate medically relevant information to the treating physician., Main Outcome Measures: Clinical relevance of whole-genome sequencing and time to communicate validated results to the treating physician., Results: Massively parallel paired-end sequencing allowed identification of a cytogenetically cryptic event: a 77-kilobase segment from chromosome 15 was inserted en bloc into the second intron of the RARA gene on chromosome 17, resulting in a classic bcr3 PML-RARA fusion gene. Reverse transcription polymerase chain reaction sequencing subsequently validated the expression of the fusion transcript. Novel FISH probes identified 2 additional cases of t(15;17)-negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole-genome sequencing and validation were completed in 7 weeks and changed the treatment plan for the patient., Conclusion: Whole-genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant time frame.