The vulnerability of clinical trials to volunteer bias is under-reported. Volunteer bias is systematic error due to differences between those who choose to participate in studies and those who do not.
Methods and Results
This paper extends the applications of the concept of volunteer bias by using data from a trial of probiotic supplementation for childhood atopy in healthy dyads to explore 1) differences between a) trial participants and aggregated data from publicly available databases b) participants and non-participants as the trial progressed 2) impact on trial findings of weighting data according to deprivation (Townsend) fifths in the sample and target populations. 1) a) Recruits (n = 454) were less deprived than the target population, matched for area of residence and delivery dates (n = 6,893) (mean [SD] deprivation scores 0.09[4.21] and 0.79[4.08], t = 3.44, df = 511, p<0.001). b) i)As the trial progressed, representation of the most deprived decreased. These participants and smokers were less likely to be retained at 6 months (n = 430[95%]) (OR 0.29,0.13–0.67 and 0.20,0.09–0.46), and 2 years (n = 380[84%]) (aOR 0.68,0.50–0.93 and 0.55,0.28–1.09), and consent to infant blood sample donation (n = 220[48%]) (aOR 0.72,0.57–0.92 and 0.43,0.22–0.83). ii)Mothers interested in probiotics or research or reporting infants’ adverse events or rashes were more likely to attend research clinics and consent to skin-prick testing. Mothers participating to help children were more likely to consent to infant blood sample donation. 2) In one trial outcome, atopic eczema, the intervention had a positive effect only in the over-represented, least deprived group. Here, data weighting attenuated risk reduction from 6.9%(0.9–13.1%) to 4.6%(−1.4–+10.5%), and OR from 0.40(0.18–0.91) to 0.56(0.26–1.21). Other findings were unchanged.
Potential for volunteer bias intensified during the trial, due to non-participation of the most deprived and smokers. However, these were not the only predictors of non-participation. Data weighting quantified volunteer bias and modified one important trial outcome.
This randomised, double blind, parallel group, placebo controlled trial is registered with the International Standard Randomised Controlled Trials Register, Number (ISRCTN) 26287422. Registered title: Probiotics in the prevention of atopy in infants and children.
Increased provider and professional society participation should form the basis of ongoing and future REMS standardization discussions with the FDA to work toward overall improvement of risk communication.
To address oncology community stakeholder concerns regarding implementation of the Risk Evaluation and Mitigation Strategies (REMS) program, ASCO sponsored a workshop to gather REMS experiences from representatives of professional societies, patient organizations, pharmaceutical companies, and the US Food and Drug Administration (FDA). Stakeholder presentations and topical panel discussions addressed REMS program development, implementation processes, and practice experiences, as well as oncology drug safety processes. A draft REMS decision tool prepared by the ASCO REMS Steering Committee was presented for group discussion with facilitated, goal-oriented feedback.
The workshop identified several unintended consequences resulting from current oncology REMS: (1) the release of personal health information to drug sponsors as a condition for gaining access to a needed drug; (2) risk information that is not tailored—and therefore not accessible—to all literacy levels; (3) exclusive focus on drug risk, thereby affecting patient-provider treatment discussion; (4) REMS elements that do not consider existing, widely practiced oncology safety standards, professional training, and experience; and (5) administrative burdens that divert the health care team from direct patient care activities and, in some cases, could limit patient access to important therapies.
Increased provider and professional society participation should form the basis of ongoing and future REMS standardization discussions with the FDA to work toward overall improvement of risk communication.
Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.
Osteosarcoma cell lines and tumors have been shown to express EGFR and harbor amplifications at the EGFR locus. In this study, we further analyze the genomic features of EGFR in osteosarcoma tumors and investigate whether they correlate with PTEN expression and copy number status.
EGFR and PTEN expression were assessed by immunohistochemistry (n=28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix® 50K single nucleotide polymorphism (SNP) arrays (n=31) and fluorescence in situ hybridization (FISH) (n=27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n=34). In addition, we assessed EGFRvIII expression using RT-PCR (n=24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5–23 years) and median follow up of 4.4 years.
EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23/28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16/27 cases; 59%) showed gains at the EGFR locus using FISH – amplification in 4/27 (15%) and balanced chromosome 7 polysomy in 12/27 (44%), and 12 cases showed deletions at the PTEN locus – biallelic deletions in 4/27 (15%) and monoallelic deletion in 9/27 (33%). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected.
EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus.
childhood cancer; osteosarcoma; epidermal growth factor receptor; tyrosine kinase; EGFRvIII; PTEN; single nucleotide polymorphism array
Hospital beds are potential reservoirs of bacteria in hospitals. Preventing contamination of the bed and providing a cleaner surface should help prevent hospital-acquired infections (HAIs). Most hospital beds are cleaned between patients (terminal cleaning) using quaternary ammonia compounds (quats).
The study had two objectives: identify levels of bacterial contamination on beds (including the mattress and bed deck) and evaluate a new launderable cover.
Hospital beds on a bariatric surgery ward were randomized to either receive or not receive a launderable cover (Trinity Guardion, Batesville, IN). Bacterial counts on the surface of the mattress, the bed deck, and the launderable cover were then collected using Petrifilm™ Aerobic Count Plates (Petrifilm™, 3M™, St. Paul, MN, USA) (Petrifilm™) at three time periods (before patient use, after discharge, and after terminal cleaning). Standard hospital linen was used in all rooms.
The launderable cover (n = 28) was significantly cleaner prior to patient use than were the cleaned mattresses (n = 38) (1.1 CFU/30 cm2 vs. 7.7 CFU/30 cm2; p = 0.0189). The mattresses without launderable covers became significantly contaminated during use (7.7 CFU/30 cm2 on admission vs. 79.1 CFU/30 cm2 after discharge; p < 0.001). The mattresses with launderable covers did not become contaminated (3.0 CFU/30 cm2 on admission vs. 2.5 CFU/30 cm2 at discharge; p = 0.703). After terminal cleaning, the mattress surface contamination decreased to 12.8 CFU/30 cm2 (median 3 CFU/30 cm2; SD 7.8), but the bed deck was more contaminated (6.7 CFU/30 cm2 after discharge compared to 30.9 CFU/30 cm2 after terminal cleaning; p = 0.031).
Terminal cleaning fails to eliminate bacteria from the surface of the hospital mattress. The launderable cover provides a cleaner surface than does terminal cleaning with quats, and the cover protects the bed from contamination during use.
Quantitative sodium MRI requires accurate knowledge of factors affecting the sodium signal. One important determinant of sodium signal level is the transmit B1 field strength. However, the low signal-to-noise ratio typical of sodium MRI makes accurate B1 mapping in reasonable scan times challenging. A new phase-sensitive B1 mapping technique has recently been shown to work better than the widely used dual-angle method in low-signal-to-noise ratio situations and over a broader range of flip angles. In this work, the phase-sensitive B1 mapping technique is applied to sodium, and its performance compared to the dual-angle method through both simulation and phantom studies. The phase-sensitive method is shown to yield higher quality B1 maps at low signal-to-noise ratio and greater consistency of measurement than the dual-angle method. An in vivo sodium B1 map of the human breast is also shown, demonstrating the phase-sensitive method’s feasibility for human studies.
B1 mapping; phase-sensitive B1 mapping; sodium MRI
The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-β activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.
chondrocyte; mesenchymal cells; transglutaminase 2; glycosaminoglycan; PKA; cell differentiation
Gemtuzumab ozogamicin (GO), an anti-CD33 immunoconjugate, was combined with high dose cytarabine (HiDAC; cytarabine 3 g/m2 over 3 hours daily for 5 days) for adults with relapsed or refractory AML. HiDAC plus GO 9 mg/m2 on day 7 and 4.5 mg/m2 on day 14 was not tolerated, but HiDAC followed by GO 9 mg/m2 on day 7 was safe: 12/37 (32%) patients with relapsed AML achieved complete remission. Median overall survival was 8.9 months. No grade 4 hepatic veno-occlusive disease was observed. This regimen merits further study, both in this setting and as a remission consolidation therapy.
gemtuzumab ozogamicin; acute myeloid leukemia; cytarabine; relapse
The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle–associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.
An infectious etiology has been proposed for many human cancers, but rarely have specific agents been identified. One difficulty has been the need to propagate cancer cells in vitro to produce the infectious agent in detectable quantity. We hypothesized that genome amplification from small numbers of cells could be adapted to circumvent this difficulty. A patient with concomitant chronic lymphocytic leukemia (CLL) and polycythemia vera (PV) requiring therapeutic phlebotomy donated a large amount of phlebotomized blood to test this possibility. Using genome amplification methods, we identified a new isolate (BIS8-17) of torque teno virus (TTV) 10. The presence of blood isolate sequence 8-17 (BIS8-17) in the original plasma was confirmed by polymerase chain reaction (PCR), validating the approach, since TTV is a known plasma virus. Subsequent PCR testing of plasmas from additional patients showed that BIS8-17 had a similar incidence (~20%) in CLL (n = 48) or PV (n = 10) compared with healthy controls (n = 52). CLL cells do not harbor BIS8-17; PCR did not detect it in CLL peripheral blood genomic deoxyribonucleic acid (DNA) (n = 20). CLL patient clinical outcome or prognostic markers (immunoglobulin heavy chain variable region [IGHV ] mutation, CD38 or zeta-chain associated protein kinase 70kDa [ZAP-70]) did not correlate with BIS8-17 infection. Although not causative to our knowledge, this is the first reported isolation and detection of TTV in either CLL or PV. TTV could serve as a covirus with another infectious agent or TTV variant with rearranged genetic components that contribute to disease pathogenesis. These results prove that this method identifies infectious agents and provides an experimental methodology to test correlation with disease.
Carboplatin is the most effective drug in retinoblastoma but systemic clearance is variable in young patients. While most regimens use a flat dose, individualized targeting may provide a more adjusted systemic exposure.
Patients and Methods
We compared carboplatin doses between two groups of children with retinoblastoma that were treated using a flat dose of 560 mg/m2 or a targeted AUC of 6.5 using a modified Calvert formula.
Ninety-eight patients with retinoblastoma received a total of 576 cycles of carboplatin (median 8 cycles). Fifty patients (51%) received a fixed dose per m2, 32 (33%) received a dose based on AUC, one patient received fixed dose per kilogram, and in 15 patients a combination AUC and fixed doses was used. The median cumulative carboplatin dose (mg/m2) for patients who received 8 cycles using fixed per m2 dosing was 2151.8 (range, 1414.2 – 2852.0), compared to 1104.1 for 9 patients who received 8 cycles using Calvert dosing (range, 779.0 – 1992.7)(p<0.001). For cycles given using AUC, the median percentage of the hypothetical fixed per m2 dose was 70% (range, 48%–134%). Younger patients had larger differences. Patients receiving carboplatin based on fixed per m2 dosing were 3.0 times more likely to have a platelet transfusion (95% confidence interval, 1.3–7.3).
Carboplatin administration needs to consider the changes in renal function occurring during the first months of life. The use of a targeted AUC provides the most accurate method; however, mg per kg of body weight dosing is a very reliable alternative method.
Carboplatin; Retinoblastoma; Glomerular filtration rate
The pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH) is not fully understood. We report a patient with myelodysplastic syndrome who developed symptomatic PNH following treatment with alemtuzumab. A small PNH clone, identified prior to alemtuzumab, expanded resulting in hemolytic anemia and recurrent CNS thromboses despite anticoagulation. Remission was achieved with eculizumab and fondaparinux therapy. Alemtuzumab has been associated with the development of glycosylphosphotidylinositol negative cells, but its clinical significance has been unclear. Our case emphasizes its potential clinical importance. Future studies are necessary to expand our understanding of this rare disease entity and improve its management.
Recent advances in our approach to blood component therapy in traumatic hemorrhage have resulted in a reassessment of many of the tenants of management which were considered standards of therapy for many years. Indeed, despite the use of damage control techniques, the mortality from trauma induced coagulopathy has not changed significantly over the past 30 years. More specifically, a resurgence of interest in postinjury hemostasis has generated controversies in three primary areas: 1) The pathogenesis of trauma induced coagulopathy 2) The optimal ratio of blood components administered via a pre-emptive schedule for patients at risk for this condition, ("damage control resuscitation"), and 3) The appropriate use of monitoring mechanisms of coagulation function during the phase of active management of trauma induced coaguopathy, which we have previously termed "goal directed therapy". Accordingly, recent experience from both military and civilian centers have begun to address these controversies, with certain management trends emerging which appear to significantly impact the way we approach these patients.
Romidepsin (depsipeptide or FK228) is a member of a new class of antineoplastic agents active in T-cell lymphoma, the histone deacetylase inhibitors. On the basis of observed responses in a phase I trial, a phase II trial of romidepsin in patients with T-cell lymphoma was initiated.
Patients and Methods
The initial cohort was limited to patients with cutaneous T-cell lymphoma (CTCL), or subtypes mycosis fungoides or Sézary syndrome, who had received no more than two prior cytotoxic regimens. There were no limits on other types of therapy. Subsequently, the protocol was expanded to enroll patients who had received more than two prior cytotoxic regimens.
Twenty-seven patients were enrolled onto the first cohort, and a total of 71 patients are included in this analysis. These patients had undergone a median of four prior treatments, and 62 patients (87%) had advanced-stage disease (stage IIB, n = 15; stage III, n= 6; or stage IV, n = 41). Toxicities included nausea, vomiting, fatigue, and transient thrombocytopenia and granulocytopenia. Pharmacokinetics were evaluated with the first administration of romidepsin. Complete responses were observed in four patients, and partial responses were observed in 20 patients for an overall response rate of 34% (95% CI, 23% to 46%). The median duration of response was 13.7 months.
The histone deacetylase inhibitor romidepsin has single-agent clinical activity with significant and durable responses in patients with CTCL.
Biliary atresia (BA) is a disease of the newborn which results in obstruction of the biliary tree. The cause of BA remains unknown; however, recent studies using the murine model of biliary atresia have found that rotavirus infection of the biliary epithelial cell (cholangiocyte) triggers an inflammatory response. We hypothesized that rotavirus infection of cholangiocytes results in the release of chemokines, important mediators of the host immune response.
In vivo, Balb/c pups were injected with rhesus rotavirus (RRV) or saline, and, their extrahepatic bile ducts were microdissected 2,5, 7, and 14 days after injection. Next, an immortalized cholangiocyte cell line (mCl) was incubated with RRV or serum free media. Qualitative and quantitative chemokine assement was performed using ELISA, PCR, and immunohistchemistry.
In vivo, increased levels of the chemokines MIP-2, MCP-1, KC and RANTES were found in RRV-infected murine bile ducts. In vitro, infected mCl cells produced increasing amounts of these same chemokines in relation to dose and time.
These novel results suggest that chemokine expression by RRV-infected cholangiocytes may trigger a host inflammatory process that causes bile duct obstruction. Understanding how viral infection initiates this response may shed light on the pathogenesis of biliary atresia.
Chronic lymphocytic leukemia (CLL) represents the outgrowth of a CD5+ B cell. Its etiology is unknown. The structure of membrane Ig on CLL cells of unrelated patients can be remarkably similar. Therefore, antigen binding and stimulation could contribute to clonal selection and expansion as well as disease promotion. Initial studies suggest that CLL mAbs bind autoantigens. Since apoptosis can make autoantigens accessible for recognition by antibodies, and also create neo-epitopes by chemical modifications occurring naturally during this process, we sought to determine if CLL mAbs recognize autoantigens associated with apoptosis. In general, ~60% of CLL mAbs bound the surfaces of apoptotic cells, were polyreactive, and expressed unmutated IGHV. mAbs recognized two types of antigens: native molecules located within healthy cells, which relocated to the external cell surface during apoptosis; and/or neoantigens, generated by oxidation during the apoptotic process. Some of the latter epitopes are similar to those on bacteria and other microbes. Although most of the reactive mAbs were not mutated, the use of unmutated IGHV did not bestow autoreactivity automatically, since several such mAbs were not reactive. Particular IGHV and IGHV/D/J rearrangements contributed to autoantigen binding, although the presence and degree of reactivity varied based on specific structural elements. Thus, clonal expansion in CLL may be stimulated by autoantigens occurring naturally during apoptosis. These data suggest that CLL may derive from normal B cells whose function is to remove cellular debris, and also to provide a first line of defense against pathogens.
Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18).Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.
Biliary atresia is a devastating disorder of the newborn in which afflicted infants develop inflammation and fibrosis of the extrahepatic biliary tract, resulting in cirrhosis and end-stage liver disease. Infection with a virus is thought to be a contributing factor in the etiology of biliary atresia. In the murine model of biliary atresia, perinatal exposure to rhesus rotavirus (RRV) results in biliary epithelial cell infection causing bile duct obstruction. The purpose of this study was to determine if tropism for the biliary epithelial cell was unique to RRV. Newborn mice underwent intraperitoneal injection with five strains of rotavirus: RRV (simian), SA11-FM (simian/bovine), SA11-SM (simian), EDIM (murine), and Wa (human). RRV and SA11-FM caused clinical manifestations of bile duct obstruction and high mortality. SA11-SM caused clinical signs of hepatobiliary injury but the mortality was markedly reduced. EDIM and Wa caused no sign of hepatobiliary disease. The systemic and temporal distribution of viral protein and live virus varied according to the injected strain. Immunohistochemistry revealed that RRV and SA11-FM targeted the biliary epithelial cells. In contrast, SA11-SM was found in the liver but in not in the biliary epithelium. These results indicate that strain-specific characteristics dictate tropism for cells of hepatobiliary origin which in turn impact the ability to induce the murine model of biliary atresia.
Analyses of Ig VHDJH rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the VL and JL gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express VL genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific VL and JL segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the VH repertoire, VL gene use was not significantly different than that of normal B-lymphocytes. In addition, Vκ genes that lie more upstream on the germline locus were less frequently mutated than those at the 3′ end of the locus; this was not the case for Vλ genes and is not for VH genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.
Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.
Previous studies suggest that the diversity of the expressed variable (V) region repertoire of the immunoglobulin (Ig)H chain of B-CLL cells is restricted. Although limited examples of marked constraint in the primary structure of the H and L chain V regions exist, the possibility that this level of restriction is a general principle in this disease has not been accepted. This report describes five sets of patients, mostly with unmutated or minimally mutated IgV genes, with strikingly similar B cell antigen receptors (BCRs) arising from the use of common H and L chain V region gene segments that share CDR3 structural features such as length, amino acid composition, and unique amino acid residues at recombination junctions. Thus, a much more striking degree of structural restriction of the entire BCR and a much higher frequency of receptor sharing exists among patients than appreciated previously. The data imply that either a significant fraction of B-CLL cells was selected by a limited set of antigenic epitopes at some point in their development and/or that they derive from a distinct B cell subpopulation with limited Ig V region diversity. These shared, stereotyped Ig molecules may be valuable probes for antigen identification and important targets for cross-reactive idiotypic therapy.
Ig variable region genes; human; B lymphocytes; antibodies; autoantibodies
Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged VHDJH and VLJL genes of 25 non–IgM-producing B-CLL cases, we found five IgG+ cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb’s reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG+ B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both.
Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor α, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal-DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.
Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.
B lymphocyte; chronic lymphocytic leukemia; somatic hypermutation; Ig gene; V gene diversification
Objective: To evaluate the relative effectiveness of computer and manual reminder systems on the implementation of a clinical practice guideline.
Design: Seventy-eight outpatients in a mental health clinic were randomly assigned within clinician to one of the two reminder systems. The computer system, called CaseWalker, reminded clinicians when guideline-recommended screening for mood disorder was due, ensured the fidelity of the diagnosis of major depressive disorder to criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV), and generated a progress note. The manual system was a checklist inserted in the paper medical record.
Measures: Screening rates for mood disorder and the completeness of the documentation of which DSM-IV criteria were met by patients who were said to have major depressive disorder were compared.
Results: The CaseWalker, compared with the paper checklist, resulted in a higher screening rate for mood disorder (86.5 vs. 61 percent, P = 0.008) and a higher rate of complete documentation of DSM-IV criteria (100 vs. 5.6 percent, P < 0.001).
Conclusions: In an outpatient mental health clinic, computer reminders were shown to be superior to manual reminders in improving adherence to a clinical practice guideline for depression.