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Purpose: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders.Experimental Design: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL.Results: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them.Conclusions: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Supplementary Figure S1 from HLA Class I–Associated Immunodominance Affects CTL Responsiveness to an ESO Recombinant Protein Tumor Antigen Vaccine

e23129 Background: AI therapy causes joint pain in up to half of women, and up to 20% become non-compliant with treatment due to pain and discomfort. This pilot study investigated the efficacy of sukshma vyayama in improving AI-induced joint pain and evaluated the feasibility of delivering the intervention on Facebook. Methods: Breast cancer patients undergoing treatment with AI's with self-reported arthralgia were recruited via an IRB-approved announcement posted in two closed breast cancer support groups on Facebook to participate in a yoga study delivered on Facebook. Participants completed BPI, DASH, PRAI and WOMAC questionnaires before and after the study. Intervention consisted of 12 joint loosening exercises performed in a chair, once daily for 12 minutes, Monday-Friday for 4 weeks. Asynchronous video demonstrations were available in a secret Facebook group and viewing confirmed by typing "done" (time-stamped) in comments. Results: 200 women responded. 38 met the inclusion criteria/consent, 26 completed the online consent, interventions and pre/post questionnaires. Paired simple t tests results showed significant (P < 0.05) improvement in all the pain measures and quality of life parameters after yoga intervention compared to baseline. Conclusions: This study provides the first evidence that it is feasible to teach sukshma vyayama to patients on Facebook and that the intervention significantly improves AI-induced arthralgia. Teaching yoga via social media may provide better access to this therapeutic modality to patients at all points in the cancer care continuum globally. [Table: see text]

Follicular lymphoma (FL) is the second most common type of B-cell lymphoma in adults in the Western hemisphere. FL can occur within lymph nodes or at extranodal sites, and is typically characterize...

Purpose: ESO is a tumor-specific antigen with wide expression in human tumors of different histologic types and remarkable spontaneous immunogenicity. We have previously shown that specific TH1 and antibody responses can be elicited in patients with no detectable preexisting immune responses by vaccination with rESO administered with Montanide ISA-51 and CpG ODN 7909. The purpose of the present study was to characterize vaccine-induced ESO-specific CD4+ T cell responses. Experimental Design: We generated CD4+ T cell clones from patient C2, who had the highest CD4+ T cell response to the vaccine, and analyzed their fine specificity and HLA class II restriction to determine the recognized epitope. We then assessed the response to the identified epitope in all vaccinated patients expressing the corresponding HLA class II allele. Results: We found that ESO-specific CD4+ T cell clones from patient C2 recognize peptide ESO119-143 (core region 123-137) presented by HLA-DR52b (HLA-DRB3*0202), a MHC class II allele expressed by about half of Caucasians. Importantly, following vaccination, all patients expressing DR52b developed significant responses to the identified epitope, accounting for, on average, half of the total CD4+ T cell responses to the 119-143 immunodominant region. In addition, analysis of ESO-specific DR52b-restricted CD4+ T cells at the clonal level revealed significant conservation of T cell receptor usage among different individuals. Conclusions: The identification of a DR52b-restricted epitope from ESO that is immunodominant in the context of vaccine-elicited immune responses is instrumental for the immunologic monitoring of vaccination trials targeting this important tumor antigen.

Purpose: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. Experimental Design: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. Results: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. Conclusions: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Das Outcome nach Kreislaufstillstand und kardiopulmonaler Reanimation hangt von entscheidenden Interventionen ab, besonders fruhzeitiger Defibrillation, effektiver Thoraxkompression und erweiterten lebensrettenden Masnahmen. Die Definitionen und Berichtsschemata des Utstein-Style sind in publizierten Studien zum Kreislaufstillstand intensiv genutzt worden, und dies hat zu einem groseren Verstandnis der Elemente der praktischen Reanimation gefuhrt sowie zum Fortschritt in Richtung eines internationalen Konsenses uber Wissenschaft und Richtlinien der Reanimation. Trotz der Entwicklung der Utstein-Schemata zur Standardisierung von Forschungsberichten uber Kreislaufstillstand mussen internationale Register erst noch geschaffen werden. Im April 2002 traf sich eine Taskforce in Melbourne, Australien, um die weltweiten Erfahrungen mit den Utstein-Definitionen und -Schemata zu bewerten. Die Taskforce uberarbeitete im Konsens das hauptsachliche Berichtsschema und die Definitionen. Es wurde Wert darauf gelegt, auf vorhandenen Definitionen aufzubauen sowie Datenelemente und operationale Definitionen nur zu andern, wenn publizierte Daten und Erfahrungen aus Registern, die den Utstein-Style verwendet hatten, dafur eine Grundlage boten. Das Augenmerk lag darauf, die Komplexitat bestehender Schemata zu verringern und logistische Schwierigkeiten bei der Erhebung spezieller Kern- oder zusatzlicher Daten (d. h. essenziell und wunschenswert), die von fruheren Utstein-Konsensuskonferenzen empfohlen worden waren, zu berucksichtigen. Ebenso befasste man sich mit Widerspruchen zwischen der Terminologie von innerklinischen und auserklinischen Utstein-Schemata. Die Taskforce hat ein Hilfsmittel zur Darstellung essenzieller Daten erstellt, das sowohl zur Qualitatsverbesserung (Register) als auch fur Forschungsberichte verwendet werden kann und das fur Erwachsene ebenso geeignet ist wie fur Kinder. Das revidierte und vereinfachte Schema beinhaltet praktische und knappe operationale Definitionen. Es ist zu erwarten, dass das uberarbeitete Schema dazu beitragt, bessere und exaktere Berichte uber alle Kreislaufstillstande und Reanimationsversuche anzufertigen. Probleme mit Datendefinition, Erhebung, Verlinkung, Vertraulichkeit, Management und Einfuhrung von Registern werden angesprochen, und es werden mogliche Losungen angeboten. Die einheitliche Sammlung und Nachverfolgung von Registerdaten soll in jedem Krankenhaus, jedem Rettungsdienst und jeder Gemeinde eine kontinuierliche Qualitatsverbesserung ermoglichen.

Outcome following cardiac arrest and cardiopulmonary resuscitation is dependent on critical interventions, particularly early defibrillation, effective chest compressions, and advanced life support. Utstein-style definitions and reporting templates have been used extensively in published studies of cardiac arrest, which has led to greater understanding of the elements of resuscitation practice and progress toward international consensus on science and resuscitation guidelines. Despite the development of Utstein templates to standardize research reports of cardiac arrest, international registries have yet to be developed. In April 2002 a task force of ILCOR met in Melbourne, Australia, to review worldwide experience with the Utstein definitions and reporting templates. The task force revised the core reporting template and definitions by consensus. Care was taken to build on previous definitions, changing data elements and operational definitions only on the basis of published data and experience derived from those registries that have used Utstein-style reporting. Attention was focused on decreasing the complexity of the existing templates and addressing logistical difficulties in collecting specific core and supplementary (i.e., essential and desirable) data elements recommended by previous Utstein consensus conference. Inconsistencies in terminology between in-hospital and out-of-hospital Utstein templates were also addressed. The task force produced a reporting tool for essential data that can be used for both quality improvement (registries) and research reports and that should be applicable to both adults and children. The revised and simplified template includes practical and succinct operational definitions. It is anticipated that the revised template will enable better and more accurate completion of all reports of cardiac arrest and resuscitation attempts. Problems with data definition, collection, linkage, confidentiality, management, and registry implementation are acknowledged and potential solutions offered. Uniform collection and tracking of registry data should enable better continuous quality improvement within every hospital, EMS system, and community.

With the strong encouragement of leading health care agencies, business principles are being implemented throughout health care, including emergency medical services (EMS). The reason is simple--quality of care can be enhanced by incorporating the management concepts of continuous quality improvement (CQI). The CQI process couples carefully identified, measurable performance indicators with information systems to monitor, analyze, and trend data. Benchmarking outcomes with other EMS systems allows the identification of "best practices" and the evolution of standards. Emergency medical services professionals must actively participate with the broader health care community in creating performance measurements to ensure that high-quality care is delivered consistently.

Celiac disease (CD) is underdiagnosed, and iron-deficiency anemia (IDA) is a common presentation of CD. No guidelines exist in the literature for screening for CD among those with IDA in the United States. We surveyed hematologists to deter- mine rates of CD screening in patients with IDA.A survey was e-mailed to members of the American Society of Hematology.There were 385 complete responses from 4551 e-mails. Most respondents were practicing clinicians (74%), clinical researchers (10%), or laboratory researchers (6%). Specialists in benign hematology accounted for 45% of respondents, oncologists accounted for 33%, and specialists in malignant hematology accounted for 22%. The most common practice types were university-affiliated hospital (43%), private clinic (29%), community hospital (12%), and Veterans Affairs or military hospital (9%). Only 8.6% believed all patients with IDA should be screened for CD. Respondents who had completed their fellowship within 5 years were more likely than more experienced clinicians to believe that all patients with IDA should receive CD screening (OR, 2.8; CI; 1.1-7.5; P=.04). Having a higher volume of IDA patients per month also increased the likelihood of testing (P=.01). In multivariate analysis, specialists in malignant hematology (OR, 3.2; CI, 1.1-9.5; P=.04) and oncologists (OR, 3.5; CI, 1.3-9.5; P=.02) were more likely than specialists in benign hematology to screen all patients for CD, as were those who saw predominately pediatric patients with IDA vs adult patients (OR, 16.9; CI, 3.0-97.0; P=.002).Practicing hematologists infrequently screen for CD in IDA. Physicians who have recently finished their fellowship and those who see a high volume of patients with IDA are more likely to screen for CD.

Approximately 1,000 people in the United States suffer cardiac arrest each day, most often as a complication of acute myocardial infarction (AMI) with accompanying ventricular fibrillation or unstable ventricular tachycardia. Increasing the number of patients who survive cardiac arrest and minimizing the clinical sequelae associated with cardiac arrest in those who do survive are the objectives of emergency medical personnel. In 1990, the American Heart Association (AHA) suggested the chain of survival concept, with four links--early access, cardiopulmonary resuscitation (CPR), defibrillation, and advanced care--as the way to approach cardiac arrest. The recently published International Resuscitation Guidelines 2000 of the AHA have addressed advances in our understanding of the chain of survival. While the chain of survival concept has withstood a decade of scrutiny, there are only a few scientifically rigorous research studies that support changes in prehospital patient care. Additional research efforts carried out in the prehospital setting are needed to support the concepts included in the chain of survival for cardiac arrest patients. Participants at the second Turtle Creek Conference, a meeting of experts in the field of emergency medicine held in Dallas, Texas, on March 29-31, 2000, discussed these and other issues associated with prehospital emergency care in the cardiac arrest patient. This paper addresses a number of the issues associated with each of the links of the chain of survival, the evidence that exists, and what should be done to achieve the clinical evidence needed for true clinical significance. Also included in this paper are the consensus statements developed from small discussion groups held after the main presentation. These comments provide another perspective to the problems and to possible approaches to deal with them.

Despite its long history and current prominence in U.S. communities, only limited data describe the national characteristics of emergency medical services (EMS) care in the United States. We sought to characterize out-of-hospital EMS care in the United States.We conducted an analysis of the 2010 National Emergency Medical Services Information System (NEMSIS) research data set, encompassing EMS emergency response data from 29 states. From these data, we estimated the national number and incidence of EMS responses. We also characterized EMS responses and the patients receiving care.There were 7,563,843 submitted EMS responses, corresponding to an estimated national incidence of 17.4 million EMS emergency responses per year (56 per 1,000 person-years). The EMS response incidence varied by U.S. Census region (South 137.4 per 1,000 population per year, Northeast 85.2, West 39.7, and Midwest 33.3). The use of lights and sirens varied across Census regions (Northeast 90.3%, South 76.7%, West 68.8%, and Midwest 67.5%). The percentage of responses resulting in patient contact varied across Census regions (range 78.4% to 95.7%). The EMS time intervals were similar between Census regions; response median 5 minutes (interquartile range [IQR] 3-9), scene 14 minutes (10-20), and transport 11 minutes (7-19). Underserved populations (the elderly, minorities, rural residents, and the uninsured) were large users of EMS resources.These data highlight the breadth and diversity of EMS demand and care in the United States.

Objective: Prior studies describe airway management by single EMS agencies, regions or states. We sought to characterize out-of-hospital airway management interventions, outcomes and complications across the United States. Methods: Using the 2008 National Emergency Medical Services Information System (NEMSIS) PublicRelease Data Set containing data from 16 states, we identified patients receiving advanced airway management, including endotracheal intubation (ETI), alternate airways (Combitube, Laryngeal Mask Airway (LMA), King LT, Esophageal-Obturator Airway (EOA)), and cricothyroidotomy (needle and open). We examined airway management success and complications in the full cohort and in key subsets (cardiac arrest, non-arrest medical, non-arrest injury, children

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

After liver transplantation, reinfection of the newly engrafted liver with hepatitis C virus is essentially universal in patients who are viremic at the time of transplantation. Treatment with interferon preparations with or without ribavirin is recommended in patients with marked histologic injury; however, hematologic toxicity associated with therapy has been reported, which is usually treated with growth factor support, including erythropoietin analogues. We present the first reported case of anti-erythropoietin antibody-mediated pure red cell aplasia arising in the setting of hepatitis C virus therapy in a patient who underwent living donor liver transplantation.

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8 + T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4 + Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8 + T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8 + T cells, indicated that elicitation of NY-ESO-1-specific CD8 + T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

e18025 Background: Using a chemosensitivity screening assay, we showed that combination decitabine and thioguanine was active in primary leukemia cells from patients with relapsed/refractory acute ...

Outcome after cardiac arrest and cardiopulmonary resuscitation is dependent on critical interventions, particularly early defibrillation, effective chest compressions, and advanced life support. Utstein-style definitions and reporting templates have been used extensively in published studies of cardiac arrest, which has led to greater understanding of the elements of resuscitation practice and progress toward international consensus on science and resuscitation guidelines. Despite the development of Utstein templates to standardize research reports of cardiac arrest, international registries have yet to be developed. In April 2002, a task force of the International Liaison Committee on Resuscitation (ILCOR) met in Melbourne, Australia, to review worldwide experience with the Utstein definitions and reporting templates. The task force revised the core reporting template and definitions by consensus. Care was taken to build on previous definitions, changing data elements and operational definitions only on the basis of published data and experience derived from those registries that have used Utstein-style reporting. Attention was focused on decreasing the complexity of the existing templates and addressing logistical difficulties in collecting specific core and supplementary (ie, essential and desirable) data elements recommended by previous Utstein consensus conferences. Inconsistencies in terminology between in-hospital and out-of-hospital Utstein templates were also addressed. The task force produced a reporting tool for essential data that can be used for both quality improvement (registries) and research reports and that should be applicable to both adults and children. The revised and simplified template includes practical and succinct operational definitions. It is anticipated that the revised template will enable better and more accurate completion of all reports of cardiac arrest and resuscitation attempts. Problems with data definition, collection, linkage, confidentiality, management, and registry implementation are acknowledged and potential solutions offered. Uniform collection and tracking of registry data should enable better continuous quality improvement within every hospital, emergency medical services system, and community.

As both fludarabine and rituximab are active against indolent lymphoproliferative disorders, we have studied the combination of fludarabine and rituximab in patients with low-grade lymphoma and chronic lymphocytic leukemia (CLL) in phase I/II fashion. Of 33 patients enrolled, 21(63.6%) had low-grade lymphoma and 12 (36.4%) had CLL. They received fludarabine 30 mg/m2 on days 1-4 and rituximab 125, 250 or 375 mg/m2 on day 5 at intervals of 28 days to a maximum of 8 cycles. Three patients were removed from the study because of rituximab-associated anaphylaxis and four because of prolonged hematopoietic toxicity. Toxicity and responsiveness did not differ at the different dose levels of rituximab. For 29 evaluable patients, responses were seen in 82.8% and complete responses in 34.5%. Of 7 responding patients not referred for stem cell transplantation, 6 remain in complete remission at a median follow-up of 16 months (range 4-30 months). Of 13 previously untreated patients, all responded and 46.2% had a complete response. Of 16 previously treated patients, 68.5% responded and 25% had a complete response. The combination of fludarabine and rituximab has major activity and acceptable toxicity in patients with low-grade lymphoma and CLL.

Cardiopulmonary resuscitation (CPR) involving manual external chest compression combined with artificial respiration was first described in 1960 by Kouwenhoven et al. (Kouwenhoven W, Jude JR, Knickerbocker GG. Closed-chest cardiac massage. JAMA. 1960; 173:1064-7). In the four decades since then, there have been no widely accepted alternatives for this technique. Even with the subsequent worldwide adoption of CPR and other advanced cardiac life support measures, long-term survival after prehospital cardiac arrest is still typically only 5%, to 10%. The performance of CPR must therefore be improved to increase the rate of long-term survival. Currently under development are new, alternative techniques such as interposed abdominal compression (IAC), active compression-decompression (ACD), pneumatic and nonpneumatic circumferential chest compression, and minimally invasive cardiac massage. Many of these newer techniques, compared with standard manual CPR, appear to provide superior vital organ blood flow and increased blood pressure. To date, only IAC (in-hospital only) and ACD have been shown to improve long-term survival in clinical studies. Circumferential chest compression and minimally invasive cardiac massage, on the other hand, have not yet been adequately tested in large clinical trials. Despite the difficulty and expense in studying these CPR techniques, additional research is necessary to evaluate their effectiveness in improving survival after sudden cardiac arrest.

Progressive multiple myeloma may manifest features of 'de-differentiation', including a plasmablastic appearance, failure to secrete paraprotein, extramedullary involvement, and resistance to treatment. A 44-year-old woman with kappa-light chain myeloma underwent allogeneic stem cell transplantation (SCT). Twenty months later she developed paraspinal plasmablastic myeloma in the absence of paraprotein in urine or myeloma in the marrow. The paraspinal masses responded to chemotherapy. At 30 months she developed myelomatous meningitis, which proved resistant to intrathecal chemotherapy, irradiation, and donor lymphocyte infusion (DLI). The leptomeningeal disease led to death at 38 months. This is the first report of leptomeningeal relapse of myeloma after allografting.

Since the early 1970s, various publications and legislation have contributed to the development of emergency medical services (EMS) information systems and databases. Yet, even today, EMS systems vary in their ability to collect patient and systems data and to put these data to use. In addition, no means currently exists to easily link disparate EMS databases to allow analysis at local, state, and national levels. For this reason, the National Association of State EMS Directors is working with its federal partners at the National Highway Traffic Safety Administration (NHTSA) and the Trauma and EMS program of the Health Resources and Services Administration's (HRSA's) Maternal and Child Health Bureau to develop a national EMS database. Such a database would be useful in developing nationwide EMS training curricula, evaluating patient and EMS system outcomes, facilitating research efforts, determining national fee schedules and reimbursement rates, and providing valuable information on other issues related to EMS care.

We report a case of high grade lymphoma which appeared at the site of prior injections of medications into the shaft of the penis for erectile dysfunction. We discuss the possible mechanisms of causation for this unusual form of lymphoma.

7031 Background: Rigosertib, a novel small molecule inhibitor of PI3-Kinase and PLK pathways is currently evaluated (IV infusions) in a phase III trial in higher risk MDS patients who have failed hypomethylating agents. A previous phase I study found good bioavailability and activity of orally administered rigosertib in transfusion-dependent MDS patients (ASH 2011). Here we report preliminary results from an ongoing phase II study. Methods: This is a randomized, two-arm study of oral rigosertib (560 mg bid) administered either intermittently (2 out of 3 weeks) or continuously. Transfusion-dependent patients must have received at least 4 units RBC transfusions over 8 weeks before randomization, and can receive transfusions and erythrocyte stimulating agents (ESAs) while on study. Results: Twenty nine MDS patients (25 intermediate-1 and 4 low risk per IPSS classification) have been randomized as of December 17, 2012. Overall oral rigosertib was well tolerated except for a high incidence (5 of 9 patients) of grade 2+ urinary side effects (dysuria, hematuria, cystitis, and urinary urgency), in the continuous dosing arm. Accordingly, the protocol was amended to allow all patients to be treated with intermittent dosing, with option of dose interruption/reduction resulting in a much lower frequency of urinary side effects (4/20 patients with urinary grade 2+ toxicity). Fifteen patients (none of them with del5q cytogenetic) have been treated with intermittent dosing for at least 8 weeks. Seven (47%) patients achieved transfusion independence (no RBC transfusion for at least 8 consecutive weeks), which lasted 8 to 27 + weeks. Six of 7 responding patients were refractory to prior treatment with ESAs and 5 of these 7 patients received concomitant ESAs, suggesting an effect of rigosertib on ESA resistance. Conclusions: Preliminary results of this phase II study indicate that intermittent dosing of rigosertib administered orally is well tolerated and active in producing transfusion independence in approximately 50% transfusion dependent, lower risk MDS patients. The contributing role of rigosertib and ESAs in these transfusion responses is being investigated. Clinical trial information: NCT01584531.

Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clone (NY 1) has a 15.4 kb insert by virtue of an extra 1.4 kb Eco RI fragment at its 5' most end. This clone also has a Kpn I site not present in the other two suggesting it is the product of the gamma gene on the opposite chromosome. Restriction analysis of the three clones indicates that the G gamma and A gamma genes are linked on a single continuous piece of DNA and are separated by 3.5 kb and each contains at least one large intervening sequence of 0.85 kg between the Bam HI and Eco RI sites. These findings in cloned DNA provide direct evidence for linkage and organization of the gamma genes in man.

We have analyzed human cellular DNA for its delta--and beta-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with 32P--beta-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3' end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the delta and beta genes and those containing sequences flanking either 5' to the delta gene or 3' to the beta gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, delta--and beta-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.

Sickle cell anemia (SS) is associated with abnormalities of the red cell membrane and decreased red cell deformability. The present study assesses globin chain binding to stroma in SS, sickle cell trait (AS), and nonsickling (AA) cells. The results indicate that there is preferential binding of newly synthesized beta(S) globin to red cell stroma in SS cells and preferential binding of beta(S) to stroma compared to beta(A) in AS cells. These studies show that beta(S) globin binding to stroma accompanies the membrane abnormalities in SS and AS patients.

Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [ 32 P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With Eco RI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained β-like gene sequences assayed with β globin cDNA probe. One β-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three β gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing β-like genes were absent from δβ thalassemic DNA and one new fragment containing β-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of β-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with δβ thalassemia.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.

Restriction endonuclease mapping of cellular DNA has provided a detailed map of the γ, δ, and β globin genes in man. This technique already provides the basis for the prenatal diagnosis of sickle cell anemia and some of the thalassemias by analysis of amniocentesis fluid cell DNA. In δβ thalassemia, certain cases of β° thalassemia and the α thalassemias, deletion of specific globin gene-containing DNA fragments can be used to establish a diagnosis using fetal DNA. In sickle cell anemia, a polymorphism associated with the sickle cell βs gene can be used to diagnose SS disease in 25 to 50% of fetuses at risk. Extension of this technology can be expected to result in the precise diagnosis of β+ and β° thalassemia and other cases of sickle cell anemia as well as in the near future.