By V. Anog. Air Force Institute of Technology.

Ineffective erythropoiesis beta-thalassemia major: a longitudinal study 30 gm acticin free shipping. Freeman2 1Department of Medical & Molecular Genetics order acticin 30 gm on-line, King’s College London School of Medicine buy acticin 30gm with visa, London, United Kingdom; and 2Department of Clinical Immunology, University of Birmingham Medical School, Edgbaston, Birmingham, United Kingdom The past 40 years have witnessed major advances in defining the cytogenetic aberrations, mutational landscape, epigenetic profiles, and expression changes underlying hematological malignancies. Although it has become apparent that acute myeloid leukemia (AML) is highly heterogeneous at the molecular level, the standard framework for risk stratification guiding transplant practice in this disease remains largely based on pretreatment assessment of cytogenetics and a limited panel of molecular genetic markers, coupled with morphological assessment of bone marrow (BM) blast percentage after induction. However, application of more objective methodology such as multiparameter flow cytometry (MFC) has highlighted the limitations of morphology for reliable determination of remission status. Moreover, there is a growing body of evidence that detection of subclinical levels of leukemia (ie, minimal residual disease, MRD) using MFC or molecular-based approaches provides powerful independent prognostic information. Consequently, there is increasing interest in the use of MRD detection to provide early end points in clinical trials and to inform patient management. However, implementation of MRD assessment into clinical practice remains a major challenge, hampered by differences in the assays and preferred analytical methods employed between routine laboratories. Although this should be addressed through adoption of standardized assays with external quality control, it is clear that the molecular heterogeneity of AML coupled with increasing understanding of its clonal architecture dictates that a “one size fits all” approach to MRD detection in this disease is not feasible. However, with the range of platforms now available, there is considerable scope to realistically track treatment response in every patient. Although cytogenet- Learning Objectives ics continues to provide the framework for risk stratification used to ● Consideration of technical aspects of flow cytometry and guide the management of AML, there has been inconsistency in the molecular-based approaches to minimal residual disease classification systems employed by different trial groups, with (MRD) detection in acute myeloid leukemia (AML). After advances in sequencing technology that led to the discovery of ● Appreciation of the assays that are now ready for clinical a number of novel recurrent mutational targets in AML, there has implementation and those which are currently investigational. It is reprinted with permission from Blood 2014, Volume 124. The prognostic impact of MFC-MRD is strong enough to Although pretreatment characterization of AML based on cytoge- have emerged despite study differences in the MFC assays and the netic analysis and molecular profiling can evidently distinguish limitations of now-outdated restricted antibody panels. Its clinical broad subgroups of patients with relatively favorable, unfavorable, value as a biomarker to inform therapy is therefore difficult to or intermediate prognosis, major drawbacks are that it lacks the ignore, particularly with its applicability to the majority of patients. Because replica- to reliably identify those most likely to benefit from early transplan- tion of this requires the organization of specialized core laborato- tation. Importantly, although cytogenetics and molecular profiling ries, the implementation of flow cytometric measurement of MRD can provide a snapshot of the genetic makeup of each case of has lagged behind that of real-time quantitative PCR. These may in some instances reflect differences in the cellular Assay approaches origins of the leukemia and sensitivity of the leukemic clone to Continued improvements in multiparameter flow technology and therapy, which may be influenced by the biology of the neoplastic reagents enable simultaneous measurement of up to 20 antigens/ cells, interaction with the marrow microenvironment, and interpa- markers per cell compared with 3 in initial AML MFC-MRD reports,16 allowing higher resolution detection per tube of BM cells. This capability is potentially further increased by mass cytometry (up to 45 parameters, but with slower cell acquisition). Visualiza- Despite the major advances in understanding the molecular patho- tion and analysis of immunophenotypic data may be transformed in genesis of AML, assessment of bone marrow (BM) morphology the near future by clinical use of automated analysis algorithms such as SPADE17 and ViSNE18 but at present, defining and detecting remains the standard approach to gauge treatment response in routine care and in the clinical trial setting, and is used to inform abnormal leukemic cells from the data produced by flow cytometry decisions concerning allogeneic transplantation. However, determi- still rely on manual inspection of cells by 2 parameters at a time on nation of blast percentage by light microscopy is hampered by biaxial plots with multiple gating steps of selected cell populations. Indeed, a recent This MFC-MRD analysis becomes more complex with increased study conducted in a large cohort of pediatric AML patients (n numbers of simultaneous fluorochrome parameters (ie, colors), as 203) reported marked discrepancies in evaluation of remission do the technical issues (eg, compensation, fluorescence overlap status as determined by standard morphology and flow cytometry. Conversely, 67% of patients classified on morphological grounds to have a partial There are 2 main analysis strategies for detecting MFC-MRD. The response (5% to 15% blasts) and 26% of those classified as having first, using a screening antibody panel (Figure 1), selects antigen resistant disease ( 15% blasts) actually had an excellent response combinations at diagnosis (termed leukemia-associated immunophe- according to flow cytometry, with no detectable minimal residual notypes [LAIPs]), each displayed by at least 5% to 10% of leukemic disease (MRD). Subsequent MFC-MRD monitoring tracks previously, together with the problems in using morphology to these diagnostic LAIPs (sometimes using tailored antibody combi- reliably assess remission status, there is clearly a very strong nations), with a cluster of 20 cell events in the LAIP gate being rationale for use of MRD detection methods to provide a more potentially sufficient to identify MRD—resulting in a maximum objective assessment of treatment response to develop a more sensitivity of between 10 4 and 10 5 when 500 000 nucleated cells individualized approach to management. Identifying specific LAIPs at diagnosis may be the various platforms available at present or in the near future, take improved by incorporation of more colors, thereby allowing the account of their relative advantages and limitations, and discuss the addition of further simultaneous markers required to define an most informative approach depending on the subtype of AML and abnormal cell. However, this is a double-edged sword because clinical scenario, recognizing the challenges for the laboratory instability of even 1 of the LAIP markers after treatment increases involved in the successful delivery of MRD-directed therapy. Experience of which phenotypic Hematology 2014 223 Figure 1. Outline of antibody groups used in panels for identification of AML-aberrant immunophenotypes (both for LAIP and “different- from-normal” approaches) and subsequent residual disease monitoring. Core markers are those selected for the backbone of the panel to identify myeloid blast populations. These are combined with markers from lymphoid/myelomonocytic maturation groups (megakaryocytic markers and NG2 [for MLL-rearranged AML] are not included but are more useful in pediatric AML). A stem cell combination may be included (as in the United Kingdom [UK] National Cancer Research Institute [NCRI] AML trial panel) to detect potential immunophenotypic LSC. Most sensitive, robust, aberrant phenotypes include cross-lineage expression and CD34 human leukocyte antigen (HLA) DR weak/negative. Some aberrant phenotypes may be sensitive (by testing detection by serial dilution in normal marrow) but less stable/useful in follow-up samples. Blue, antibody marker; bold blue, marker in NCRI AML trial panel; red, type of aberrant phenotype potentially detected by marker group. These may result from the phenotypic changes discussed The second analysis approach, initially validated by the Children’s previously or emergence of initially minor subpopulations (less than Oncology Group,9,20 circumvents the problems of false negatives 5% to 10% of blasts at presentation) excluded by the LAIP assay or from phenotypic shifts and emerging subclones by using a fixed may reflect chemoresistant leukemic cells that lack sufficiently antibody panel (also based on combinations in Figure 1) and an specific immunophenotypic aberrancies, even if these are identified analysis that screens for established immunophenotypic profiles to in other leukemic subpopulations. Although extensive antibody distinguish abnormal leukemic cells (including more mature sub- panel screens increase the chances of detecting rarer aberrancies, populations) from normal cells irrespective of diagnostic leukemic cost-effectiveness and sample size are practical considerations immunophenotype.

It is of note that there is no integrase in human cells so selective inhibition of this enzyme that does not induce side effects seems possible purchase 30 gm acticin with visa. There are at least four steps leading to the integration of viral DNA (Review: Lataillade 2006) buy discount acticin 30 gm on-line. All these steps may be theoretically inhibited by different integrase inhibitors buy acticin 30gm with visa. Binding of the integrase enzyme to viral DNA within the cytoplasm. This results in a stable viral DNA-integrase binding complex (pre-integration complex, PIC). This step can be inhibited by binding inhibitors such as pyrano-dipyrimides. The integrase removes a dinucleotide at each end of the viral DNA producing new 3’ hydroxyl ends within the PIC. This step can be inhibited by 3’ processing inhibitors such as diketo acids. After the transport of the PIC from the cytoplasm through a nuclear pore into the cell’s nucleus, integrase binds to the host chromosomal DNA. By doing this, integrase mediates irreversible binding of viral and cellular DNA. This step can be inhibited by integrase strand transfer inhibitors (INSTIs). The combination of viral and cellular DNA is a gapped intermediate product. The gap repair is done by host cell DNA repair enzymes. Integrase seems not to be necessary in this last step, which can be inhibited by gap repair inhibitors such as methylxanthines. For almost a decade, the development of integrase inhibitors was slow. This was largely because of a lack of good lead compounds and reliable in vitro screening assays that incorporate each of the integration steps (Lataillade 2006). Only after 2000 did development progress and the principle of strand transfer was elucidated (Hazuda 2000). Since 2005, numerous clinical studies have evaluated integrase inhibitors (mainly strand transfer inhibitors). In December 2007, raltegravir was licensed as the first integrase inhibitor for the treatment of HIV+ patients. Today, three integrase inhibitors are on the market, namely raltegravir, elvitegravir and dolutegravir. Given the good tolerability and the high potency of this drug class, INSTIs now play a major role in HIV medicine. As with other antiretroviral drug classes, however, some questions remain unan- swered. Although very well-tolerated during the first years of therapy, little is known about long-term toxicity of integrase inhibitors. There is no experience with long- term use beyond 5–10 years. Genetic resistance barriers, relatively low with ralte- gravir and elvitegravir, may also be an important issue. Increased viral rebound rates were observed with treatment-experienced patients on boosted PIs (viral load below the limit of detection) when switching to raltegravir, especially in those with pre- existing resistance (Eron 2009). As soon as integrase inhibitor resistance develops, the agent should be stopped. This way, further resistance mutations (Wirden 2009) can be avoided, as well as unnec- essary costs. Problems also exist with the measurement of plasma levels (Cattaneo 2012). Individual agents Dolutegravir (DTG, Tivicay, also part of Triumeq) is an integrase inhibitor that initially emerged via Shionogi cooperating with GSK and is now being developed by ViiV Healthcare. As a second-generation integrase inhibitor it shows improvements, 6. Overview of antiretroviral agents 101 especially with regard to pharmacokinetics (once daily unboosted administration, independent from food intake) and resistance profile. In a Phase IIa study with 35 patients, a reduction of 2. ART-naïve patients: after the encouraging results of SPRING-1, a Phase IIb study (Stellbrink 2012), dolutegravir was tested against its competitor drug, the first-in- class INSTI raltegravir. In SPRING-2, 822 patients received two NRTIs and either 50 mg QD or raltegravir 400 mg BID. At 96 weels, 81% versus 76% had achieved an unde- tectable viral load, respectively.

Widespread human epidemics have been lim- ited to H1N1 buy acticin 30gm, H2N2 cheap 30gm acticin free shipping, and H3N2 purchase 30 gm acticin otc, although occasional transfers of other subtypes occur from birds or mammals to humans. Pigs harbor H1 and H3, whereas horses have H3 and H7. Other mammals and nonaquatic birds occasionally become infected, but do not appear to maintain stable lineages over time. Influenza HA and NA molecules mediate viral attachment and entry to host cells and release of progeny viral particles by budding through themembrane of infected cells (Lamb and Krug 2001). Current under- standing assigns adsorption and entry functions to HA (Steinhauer and Wharton 1998) and release of progeny to NA (Colman 1998). However, the HA and NA molecules may have multiple active sites and various functions, and the different subtypes of each molecule differ signifi- cantly (Lamb and Krug 2001). With those caveats, a brief summary of structure and function follows. The HA mole- cule then cleaves into two parts, the terminal HA1 and the basal HA2 fragments. Cleavage exposes on the surface of HA2 a highly conserved, hydrophobic region that mediates fusion and entry via the host mem- brane (Wilson and Cox 1990; Skehel and Wiley 2000). EXPERIMENTAL EVOLUTION: INFLUENZA 209 B Receptor binding site 155 193 188 A 133 186 126 220 144 145146 226 217 D 143 205 122 208 78 E 83 54 275 53 C 278 Figure 13. This drawing is based on structural analysis of H3 hemagglutinin. Inference about other HA subtypes depends on presumed structural similarity with H3. Labeled amino acid num- bers for HA1 are a subset of the variable sites listed in Wiley et al. The sialic acid binding site oc- curs near the tip of HA1. The letters A–E locate the major regions for 210 CHAPTER 13 Tyr 195 Leu 194 Glu 190 Thr 155 His 183 HO N OH N H H O N HO 9 OH Trp 153 7 CH 8 3 O 5 NH OH 6 OH H RO 3 4 O 2 O O O Leu 226 135 O HO 225 Ser 136 224 H Tyr 98 N 137 138 98 Y→F 5 183 H→F 12 225 G→D 53 136 S →A 30 183 H→A — 225 G→R 136 136 S →T 45 190 E →A 125 226 L →P 42 153 W→A — 194 L →A 3 228 S →G 112 153 W→F 39 195 Y→F 36 Figure 13. The listing below shows the binding affinities for sialic acid when particular amino acids are changed ex- perimentally by site-directed mutagenesis (Martín et al. The number on the left defines the amino acid site in HA1, x → y shows the original and new amino acid, and the number on the right is the binding affinity of the mutant as a percentageof the affinity of the wild type. Dashesshow cases in which the recep- torsiteisnot properly expressed. Redrawn from Skehel and Wiley (2000), with permission from the Annual Review of Biochemistry, www. The sialic acid has been displaced slightly to show the structure of the fit. The amino acids numbered within and around the binding site provide a reference for the location of important residues. The bottom of the figure shows the effect on binding affinity to sialic acid caused by experimental change of particular amino acids. This space-filling model has roughly the same orientation as the schematic diagram in figure 13. Antibodies bound to HA can neutralizeinfluenza infectivity by physi- cally obstructing the sialic acid binding site. For example, the HC19 MAb binds to HA of strain X-31 (H3 subtype),partially overlapping the sialic acid binding site (Bizebard et al. The specific antibody-epitope re- gion of direct contact covers 1250 Å2,including amino acids 134, 136, 212 CHAPTER 13 153, 155, and 194. The de- pression extends 315 Å2,ofwhichtheantibody binding region covers 167 Å2. Antibody escape mutants map to the ridge of amino acids that ring the conserved amino acids in the binding pocket. Each upper arm forms an Fab frag- ment, with the binding region on the tip of the fragment. An antibody molecule can be cleaved to release two identical Fab fragments, each containing a binding region. However, other antibody escape mutants map to regions of HA away from the sialic acid binding site. Those sites are too far away to allow overlap of the direct antibody- epitope binding region with the sialic acid binding site. The Fab fragment of HC45 bound to its epitope with approximately the same kinetics as HC19 bound to its epitope, but HC19 was an order of mag- nitude more efficient at neutralization. Presumably this occurs because the Fab of HC19 causes greater obstruction of binding to sialic acid than does the more distantly bound Fab of HC45. By contrast, the full anti- body molecules of HC19 and HC45 neutralized virus in proportion to their binding affinity for their respective epitopes. HC19 binds to the tips of HA molecules away from the viral surface; thus HC19 faces relatively little obstruction when binding to intact HA on viruses.

Proton pump inhibitors and treatment durations in longer-term studies of gastroesophageal reflux disease: Comparisons of daily treatment with intermittent or on-demand treatment Study N Diagnosis Strategy 1 Strategy 2 Strategy 3 Healed Sontag Omeprazole 20 Omeprazole 20 mg 406 erosive Placebo 1997 mg daily 3 days a week esophagitis Healed Omeprazole 20 Omeprazole 20 mg Ranitidine 300 Dent 1994 204 erosive mg daily 3 days a week mg daily esophagitis Healed Sjostedt Esomeprazole 20 Esomeprazole 20 477 erosive 2005 mg daily mg on-demand esophagitis Lansoprazole Cibor a Lansoprazole 15 Lansoprazole 30 30 mg 65 NERD 2006 mg daily mg on-demand intermittent (4 weeks) a Rabeprazole 10 Rabeprazole 10 mg Bour 2005 181 NERD mg daily on-demand Janssen a Pantoprazole 20 Pantoprazole 20 432 NERD 2005 mg daily mg on-demand Symptoms of Morgan gastroesopha Rabeprazole 20 Rabeprazole 20 mg 268 2007 geal reflux mg daily on-demand disease Symptoms of Hansen 190 gastroesopha Esomeprazole 20 Esomeprazole 20 Ranitidine 300 2005 discount acticin 30 gm online, 2 geal reflux mg daily mg on-demand mg daily 2006 disease Abbreviations: NERD buy acticin 30gm line, non-erosive reflux disease cheap acticin 30 gm free shipping. Proton pump inhibitors Page 56 of 121 Final Report Update 5 Drug Effectiveness Review Project Table 15. Remission of gastroesophageal reflux disease erosions and symptoms in longer-term studies of proton pump inhibitors: Comparisons of daily treatment with intermittent or on-demand treatment Percent of treatment group with result (daily vs. Assessment of overall satisfaction with treatment was not different between regimens in 1 222 study and final quality of life scores were also not different between groups in the other 224 study. However, the mean change in quality of life scores from baseline to 6 months was 224 significantly better in the daily treatment group compared to the on-demand group (P=0. The third study of pantoprazole 20 mg found the on-demand regimen to be noninferior to the daily regimen, based on the rates of ‘treatment failure’ defined as moderate symptoms for 3 or more days, use of > 1 dose of study medication on > 3 consecutive days, or withdrawal from 226 study due to lack of efficacy. In patients presenting with symptoms of gastroesophageal reflux disease (but with no 220, 227 endoscopic examination), 2 studies found mixed results. In a study of on-demand 228 esomeprazole, the results differ by which symptom-based outcome measure is used. Statistical analyses of the results were not undertaken in the study, but here we have used a Yates corrected chi-square test. Using an outcome of “no heartburn” at 6 months, daily therapy is superior to on- demand treatment with 72% compared with 62% (P=0. However, the percentage of patients with no regurgitation at 6 months was 78% with daily therapy and 91% with on-demand treatment (P<0. The other study found that daily treatment with rabeprazole resulted in statistically significantly more heartburn-free days (90%) compared with on-demand treatment (65%; P<0. These 2 studies also report different findings in quality of life. Again, the study of esomeprazole found the daily regimen superior to the on-demand regimen in both change from baseline in quality of life and patient satisfaction. The other study found that on-demand treatment with rabeprazole resulted in greater improvement in 220 quality of life at 6 months compared to the daily regimen. Proton pump inhibitors Page 57 of 121 Final Report Update 5 Drug Effectiveness Review Project Standard-dose proton pump inhibitor compared with H2 receptor antagonist or placebo Four studies found proton pump inhibitors to be superior to ranitidine 150 mg twice daily, 3 for prevention of relapse of healed esophagitis and 1 for prevention of recurrence of symptoms of 229,230,223, 225 gastroesophageal reflux disease. After 12 months, proton pump inhibitor therapy (pantoprazole 10 mg, 20 mg, or 40 mg and omeprazole 20 mg daily) resulted in lower relapse rates compared with ranitidine therapy. In 2 studies, more patients remained healed on pantoprazole at all doses than on ranitidine, and the rate of relapse was related to the dose of pantoprazole: Relapse occurred in 60%, 32%, and 18% of the 10 mg, 20 mg, and 40 mg groups, respectively. A second study of the same doses of pantoprazole and ranitidine found similar 230 results. During the first 12 months of maintenance treatment, healing was maintained in 78% of patients treated with pantoprazole 40 mg, 55% of patients treated with pantoprazole 20 mg, 46% of patients treated with pantoprazole 10 mg, and 21% of those treated with ranitidine. This study is planned for 3 years, but only the first 12 months have been reported so far. With omeprazole, at 12 months 89% remained in remission compared with 25% on ranitidine 225 (P<0. In those with symptoms suggestive of gastroesophageal reflux disease, 72% had relief of symptoms after 6 months of esomeprazole 20 mg daily compared with 33% taking ranitidine (statistical analysis not presented). Additionally, a study of famotidine 20 mg twice daily compared with lansoprazole 15 mg daily, both as step down therapy from lansoprazole 30 mg daily for treatment of erosive esophagitis, found the proton pump inhibitor to be superior in preventing recurrence of regurgitation and heartburn, but not dysphagia or assessment of esophagitis grade after 8 weeks 231 of maintenance treatment. Fifty percent of patients taking famotidine experienced recurrence of heartburn, and 79% experienced recurrence of regurgitation compared to 0% and 7%, respectively, with lansoprazole 15 mg daily. Comparison of esomeprazole administered orally compared to esomeprazole administered intravenously A trial conducted in 246 ambulatory patients compared esophagitis healing rates at 4 weeks in patients given esomeprazole 40 mg either orally, via intravenous injection, or via intravenous 232 infusion. Patients were randomized to either 1 week of intravenous esomeprazole (injection or infusion) followed by 3 weeks of oral esomeprazole or 4 weeks of oral esomeprazole. After 4 weeks, there was no difference in healing rates among the 3 treatment groups (approximately 80%). The frequency and type of adverse events were also similar among the treatment groups. Comparison of a reduced-dose proton pump inhibitor with an H2 receptor antagonist in children One fair-quality randomized trial compared reduced-dose omeprazole with ranitidine for longer- 233 term treatment of erosive gastroesophageal reflux disease in children (Evidence Table 6). Children who had been treated with omeprazole and shown by endoscopy to be healed after 3 months began treatment with omeprazole 0. Although no statistically significant difference was found among the groups at baseline, children in the group receiving no treatment had slightly less severe esophagitis and slightly lower symptom scores than children in the other groups. They were also slightly older at enrollment and at age of symptom onset. Follow-up Proton pump inhibitors Page 58 of 121 Final Report Update 5 Drug Effectiveness Review Project endoscopy at 3 months after the end of maintenance treatment was blinded. No statistically significant differences were seen in endoscopic or histologic grade or in symptom scores.