Search

ABSTRACTDuchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame. Indeed, three exon skipping oligonucleotides have received FDA approval for use in DMD patients. Second-generation exon skipping drugs (i.e. peptide-antisense oligonucleotide conjugates) exhibit enhanced potency, and also induce dystrophin restoration in the heart. Similarly, multiple additional antisense oligonucleotide drugs targeting various exons are in clinical development in order to treat a greater proportion of DMD patient mutations. Relatively recent advances in the field of genome engineering (specifically, the development of the CRISPR/Cas system) have provided multiple promising therapeutic approaches for the RNA-directed genetic correction of DMD, including exon excision, exon reframing via the introduction of insertion/deletion mutations, disruption of splice signals to promote exon skipping, and the templated correction of point mutations by seamless homology directed repair or base editing technology. Potential limitations to the clinical translation of the splice modulation and gene editing approaches are discussed, including drug delivery, the importance of uniform dystrophin expression in corrected myofibres, safety issues (e.g. renal toxicity, viral vector immunogenicity, and off-target gene editing), and the high cost of therapy.

In this study we report the highest resolution proteomics analysis performed in dystrophic muscle to date. The use of two mouse models of Duchenne muscular dystrophy, at three different ages, has enabled the identification of mutation-independent protein signatures associated with dystrophic muscle, and the progression of disease pathology.

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder, caused by expansion of a CTG microsatellite repeat in the 3ʹ untranslated region of the DMPK(dystrophia myotonicaprotein kinase) gene. To date, novel therapeutic approaches have focused on transient suppression of the mutant, repeat-expanded RNA. However, recent developments in the field of genome editing have raised the exciting possibility of inducing permanent correction of the DM1 genetic defect. Specifically, repurposing of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system has enabled programmable, site-specific, and multiplex genome editing. CRISPR-based strategies for the treatment of DM1 can be applied either directly to patients, or indirectly through the ex vivomodification of patient-derived cells, and they include excision of the repeat expansion, insertion of synthetic polyadenylation signals upstream of the repeat, steric interference with RNA polymerase II procession through the repeat leading to transcriptional downregulation of DMPK, and direct RNA targeting of the mutant RNA species. Potential obstacles to such therapies are discussed, including the major challenge of Cas9 and guide RNA transgene/ribonuclear protein delivery, off-target gene editing, vector genome insertion at cut sites, on-target unintended mutagenesis (e.g., repeat inversion), pre-existing immunity to Cas9 or AAV antigens, immunogenicity, and Cas9 persistence.

ABSTRACTMultiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4ºC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37ºC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from ~1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells.

ABSTRACTThis review embarks upon a cell death journey from the discovery of apoptosis and necrosis through to the coalescence of these: necroptosis. The mechanisms of 2 emerging necrotic cell death pathways, pyroptosis and ferroptosis, will be explored before delving into apoptotic and necroptotic signaling cascades, highlighting the complex interplay between molecular players. The involvement of the ripoptosome, interferon signaling and DNA damage in necroptosis will be discussed briefly. The major focus is on necroptosis initiation by tumor necrosis factor-α (TNFα) and its cognate receptor TNFR1, caspase-independent RIP1/RIP3/MLKL necrosome activation and cell death propagation by damage-associated molecular pattern (DAMP) release. Finally, the implications of a complex cell death signaling network will be revealed in the context of cancer biology and therapy. The clinical contribution of the discovery of necroptosis as an unequivocally new way of dying is monumental and could drastically alter cancer therapy strategies in the future.

The article offers information on the issues related to the development of English legal language. It discusses several terms derived from Old English which are used under terminology such as bench, oath and house in courthouse. It also mentions of vast usage of French and Latin terms in the English legal system.

We investigated a recent (January 1999 to December 2009) cohort of 95 elderly Hodgkin lymphoma subjects. At diagnosis, median age was 67 years (range, 60-89 years), whereas 61% had significant comorbidity, 26% were unfit, 17% had a geriatric syndrome, and 13% had loss of activities of daily living. Overall response rate to therapy was 85%, whereas incidence of bleomycin lung toxicity was 32% (with associated mortality rate, 25%). With 66-month median follow-up, 2-year and 5-year overall survival were 73% and 58%, respectively (advanced-stage, 63% and 46%, respectively). Most International Prognostic Score factors were not prognostic on univariate analyses, whereas Cox multivariate regression identified 2 risk factors associated with inferior overall survival: (1) age more than 70 years (2.24; 95% CI, 1.16-4.33, P = .02) and (2) loss of activities of daily living (2.71; 95% CI, 1.07-6.84, P = .04). Furthermore, a novel survival model based on number of these risk factors (0, 1, or 2) showed differential 2-year OS of 83%, 70%, and 13%, respectively (P < .0001) and 5-year OS of 73%, 51%, and 0%, respectively (P < .0001).

We investigated a recent (January 1999 to December 2009) cohort of 95 elderly Hodgkin lymphoma subjects. At diagnosis, median age was 67 years (range, 60-89 years), whereas 61% had significant comorbidity, 26% were unfit, 17% had a geriatric syndrome, and 13% had loss of activities of daily living. Overall response rate to therapy was 85%, whereas incidence of bleomycin lung toxicity was 32% (with associated mortality rate, 25%). With 66-month median follow-up, 2-year and 5-year overall survival were 73% and 58%, respectively (advanced-stage, 63% and 46%, respectively). Most International Prognostic Score factors were not prognostic on univariate analyses, whereas Cox multivariate regression identified 2 risk factors associated with inferior overall survival: (1) age more than 70 years (2.24; 95% CI, 1.16-4.33, P= .02) and (2) loss of activities of daily living (2.71; 95% CI, 1.07-6.84, P= .04). Furthermore, a novel survival model based on number of these risk factors (0, 1, or 2) showed differential 2-year OS of 83%, 70%, and 13%, respectively (P< .0001) and 5-year OS of 73%, 51%, and 0%, respectively (P< .0001).

Heat shock proteins are vital to cell survival under conditions of stress. They bind client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes and recovery of proteins from aggregates. Heat shock protein inhibitors are a diverse group of novel agents that have been demonstrated to have pro-apoptotic effects on malignant cells through inhibition of ATP binding on the ATP/ADP-binding pocket of the heat shock protein. Initial development of heat shock protein 90 inhibitors, geldanamycin and 17-AAG, were limited by hepatotoxicity and the need for solvent carrying agents. In contrast, retaspimycin, or IPI-504, a derivative of geldanamycin and 17-AAG, is highly soluble in water and generally well tolerated. In Phase I/II trials, retaspimycin has shown activity in NSCLC and gastrointestinal stromal tumor. The most promising activity was observed in gastrointestinal stromal tumors. Phase I/II trials are currently underway to evaluate the dosing schedules and activity of IPI-504 in breast cancer. Given the in vitroactivity in diffuse large B-cell lymphoma, mantle cell lymphoma, melanoma, leukemia and pancreatic cancer, current and future trials are of clinical interest. This article reviews IPI-504 and its utility in a wide variety of cancer phenotypes.

Survival rates for eHL (most commonly defined as age over 60 years) are significantly and disproportionately inferior to those achieved in younger patients (pts). Reported 5-year event-free survival (EFS) rates for advanced-stage eHL have historically ranged from 30%–45% with 5-year overall survival (OS) rates of 40%–55%. Potential explanations for these discrepant inferior outcomes include co-morbidities precluding delivery of chemotherapy, treatment-related toxicities, and biology of disease. Data examining toxicity, survival, and prognostication for eHL in the modern era are lacking.We report a multicenter collaboration that retrospectively investigated a large cohort of eHL pts treated over a recent 12-year period (June 1999 December 2010). We examined clinical characteristics, treatment-related toxicities, and outcomes. Furthermore, comorbidities were assessed using the Cumulative Illness Rating Scale for Geriatrics (CIRS-G) scale. Patients were also classified as “fit” vs “not fit” (i.e., loss of activities of daily living (ADLs), >3 grade 3 CIRS-G, any grade 4, and/or presence of geriatric syndrome). Univariate associations with survival were determined, while a multivariate Cox proportional hazards model was completed. Additionally, a prognostic model was constructed by classification and regression tree (CART) analysis.113 eligible pts were identified, while 95 had full data and were analyzed. Among these 95 pts (58M:37F), median age was 67 years (range, 60–89), with 33% of pts ages 70–79 years, while 7% were 80–89 years. 27% of pts had a prior malignancy at a median of 8.4 years (range, 1–25 years) prior to HL diagnosis, for which most had received radiation. At HL diagnosis, there were presence of B symptoms in 54%, performance status 2–4 in 27%, 21% with history of coronary artery disease, and 16% had diabetes. In terms of functional status, 61% of pts had a CIRS-G grade 3 or 4 in at least 1 category, while 46% had a cumulative score >6. Further, 17% had presence of a geriatric syndrome, 26% were classified as 'not fit', and 13% had loss of ADLs at time of HL diagnosis. 25% of pts had bone marrow involvement, while 20% had other extranodal disease (most common: bone and lung). Altogether, 64% of pts had stage III/IV disease, of which 58% had an international prognostic score (IPS) of 4–7. HL histology was nodular sclerosis in 47%, mixed cellularity 31%, NOS 16%, lymphocyte predominant 5%, and lymphocyte depleted 1%. Primary treatment consisted of: ABVD-based (n=67), MOPP-based (n=6), BCVPP (n=6), ChlVPP (n=5), radiation alone (n=4), CHOP (n=3), hospice (n=2), BEACOPP (n=1), and watchful waiting (n=1). 78% of pts received granulocyte-colony stimulating factor (G-CSF) with therapy. The overall response rate (ORR) among treated pts was 85% (73% complete remission (CR) rate). In terms of toxicity, the incidence of bleomycin lung toxicity (BLT) was 32%, which had an associated mortality rate of 25%. Notably, the incidence of BLT was 37% vs 0% among pts who received G-CSF vs not, respectively (p=0.041). With a median follow-up of 66 months (6–154) months, the 5-year EFS and OS for all eHL pts were 44% and 58%, respectively (stage I/II: 61% and 79%; and stage III/IV: 36% and 46%; p=0.009 and p=0.001, respectively). Prognostic factors that predicted survival on univariate analysis are detailed in Table 1. On multivariate regression analysis, 2 factors were associated with inferior survival: 1) age ≥ 70 years (EFS: 1.76 (95%CI 0.98–3.16), p=0.06; and OS: 2.24 (95%CI 1.16–4.33), p=0.02) and 2) Loss of ADLs (EFS: 2.47 (95%CI 0.98–6.21), p=0.055; and OS: 2.71 (95%CI 1.07–6.84), p=0.04). Furthermore, a survival model by multivariate CART analysis, based on number of these 2 adverse factors (0, 1, 2), was formed: 5-year EFS 73%, 51%, 0%, respectively (p<0.0001), and 5-year OS 93%, 68%, 11%, respectively (p<0.0001) (Figure 1).This large, retrospective multicenter analysis of eHL found a high ORR and CR rate, but poor long-term outcomes. As shown previously, a significant percentage of eHL pts suffered BLT, which was fatal in 25% and appeared strongly related to use of growth factors. Further, several geriatric specific variables, including loss of ADL's, was highly predictive of outcome and should be prospectively incorporated into clinical trials. Finally, we developed a new prognostic model for eHL that identifies pt populations with markedly divergent outcomes.No relevant conflicts of interest to declare.

The prevalence of NHL in patients (pts) ≥ 80 years has recently tripled; however, data on prognostic factors and treatment for this very elderly population is sparse.A multicenter retrospective analysis of NHL pts ≥80 years diagnosed between (1999–2009) was completed. Detailed characteristics obtained include geriatric syndromes (GS), activities of daily living (ADLs), and co-morbidities using the Cumulative Illness Rating Scale-Geriatrics (CIRS-G). Further, univariate associations with survival were determined, while a multivariate Cox regression proportional hazards model was performed.We identified 303 pts: 170 aggressive NHL (84% B-cell and 16% T-cell) and 133 indolent NHL (82% B-cell and 18% T-cell). Median age was 84 years (range 80–95). Poor performance status (PS), high LDH, and extranodal disease were more common in aggressive vs. indolent NHLs (P=0.004, 0.005, and 0.002, respectively). Furthermore, aggressive NHL pts presented more frequently with advanced stage and higher relative prognostic scores (P=0.0002 and 0.003 respectively). Loss of any ADL was more frequent in aggressive histology (P=0.002). Diffuse large B-cell NHL was the most common histology overall, while follicular lymphoma was the most diagnosed indolent NHL. At least one GS was present in 80 pts (26%) with dementia being most common (26%). Loss of at least one ADL was seen in 14%, while 30% of pts were considered “non-fit” at diagnosis. Of available treatment data in aggressive B-NHL, 62% received rituximab (R)-containing regimen and 18% received chemotherapy without R. Those with aggressive T-cell NHL were treated with systemic therapy in 74%, topical in 15%, radiation in 4%, and no therapy in 7%. Indolent B-cell NHL had observation in 38% for a median of 26 months, R alone in 21%, R plus chemotherapy in 15%, radiation alone in 7%, and chemotherapy alone in 9%. Cutaneous T-cell pts received topical therapies in 42%, systemic therapy in 29%, and radiation alone in 17%. At 49-month median follow-up, 4-year progression-free (PFS) and overall survival (OS) for aggressive NHL were 31% and 44%, respectively (stage I/II: PFS 53% and OS 66%; stage III/IV: PFS 20% and OS 32%; p<0.0001 and 0.0002, respectively). Four-year PFS and OS for indolent NHL were 44% and 66%, respectively, regardless of stage. Prognostic factors predicting OS and PFS on univariate are detailed in Table 1. Cox multivariate regression analysis identified two factors predicting inferior outcome for both NHL groups: lack of complete response (CR) (indolent: PFS, P=0.03; OS, P=0.01; aggressive: PFS, P<0.0001; OS, P<0.0002) and loss of ADLs (indolent: PFS, P=0.003; OS, P=0.0004; aggressive: PFS, P=0.002; OS, P<0.0001).To our knowledge, this represents the largest analysis of very elderly NHL reported to date. We identified that loss of ADL's and lack of CR to initial treatment were robust predictors of survival in very elderly NHL, irrespective of histology, and they superseded the prognostic value of either PS or age. Geriatric assessments, including ADLs, are powerful predictors of outcome in very elderly NHL and should be prospectively studied.No relevant conflicts of interest to declare.

Abstract 2625

Abstract 3668