Gut Toxicity Research Articles

A Pilot Study Of Continuous Infusion Mycophenolate Mofetil For Graft Versus Host Disease Prophylaxis

Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA) has been used successfully for graft-versus-host disease (GVHD) prophylaxis. Most MMF dosing regimens and pharmacokinetic (PK) parameters have been established in solid organ transplant recipients. Empiric fixed-dose-escalation strategies have failed to achieve target MPA exposure, especially in the early period following conditioning therapy. We hypothesized that a PK-based dosing approach using a novel continuous infusion (CI) method of MMF will be able to achieve and maintain target MPA exposure. The objective of this study was to evaluate the safety and feasibility of this MMF dosing approach in pediatric BMT recipients. Fifteen patients were enrolled in the study. The median age at transplant was 8.4 years (0.9 – 21.5); eight were female and seven were male. Thirteen patients were transplanted for hematologic malignancies and two patients for nonmalignant diseases. Stem cell sources included matched-sibling bone marrow in seven, 10/10 allele matched donor bone marrow in four, and unrelated cord blood in four cases. All patients received a myeloablative conditioning regimen with either cyclophosphamide-TBI (n=8) or busulfan-cyclophosphamide (n=7). In addition, of eight unrelated donor transplants, six patients received equine ATG and two received fludarabine. All patients received cyclosporine A and MMF for GVHD prophylaxis. Intravenous (IV) cyclosporine was initiated on day -2 as a continuous infusion (target blood level: 160 to 250 ng/mL). IV MMF was started on day “0” in a dose of 15 mg/kg every 8 hours. After a minimum of five doses, serial blood samples were collected within one dosing interval for the 1st PK analysis. Using these results, MMF was converted from intermittent IV to CI administration to target total MPA AUC0-24 of 40-80 mcg/mL/hr. MMF dose was adjusted to maintain a total MPA steady-state concentration (Css) of 1.7-3.3 mcg/mL. CI MMF was converted to every-8-hour oral dosing when patients were tolerating oral medications. A 2nd PK study was performed after a minimum of five oral doses, and dose adjustments were made to maintain a total MPA trough concentration (Ctrough) of 1-3.5 mcg/mL. MMF was continued until day 42, and in the absence of acute GVHD, tapered off over 8 weeks. On the initial 15 mg/kg q8 IV MMF dose schedule, MPA half-life was 1.3±1.3 hrs and AUC0-24 was 44.3±60.3 mg* hr/L. During the CI schedule, MMF dose was 44±16 mg/kg/day and MPA AUC0-24 38.4±20.7 mg* hr/L, and thirteen of fifteen (87%) patients achieved MPA Css within target of 1.7–3.3 mcg/mL. On conversion to oral route, median MMF dose was 45±19mg/kg/day, MPA half-life 1.95±2.3 hrs and AUC0-24 38.3±16.0 mg* hr/L. MPA clearance was 17±14 ml/min/kg. Higher steady state MPA concentrations were achieved with CI method (Css 1.6 ±0.9 mcg/mL) compared with intermittent IV or oral dosing (Ctrough 0.7±0.9 mcg/mL). All patients achieved neutrophil engraftment at 13 ± 11 days post-transplant, and platelet engraftment at 45 ± 72 days post-transplant. Seven out of 15 patients (47%) developed stage I-IV acute GVHD. Three patients had stage III-IV aGVHD (20%); they were steroid-refractory but went into remission with additional therapy. For 11 patients with a minimum of one year follow-up post-transplant, one (9%) developed “overlap” chronic GVHD. MMF was discontinued without weaning in four patients: in three of them due to concern of delay in blood count recovery and all of them achieved hematopoietic engraftment, and in one patient with severe aGVHD due to potential concern for MMF gut toxicity. One patient with severe congenital neutropenia developed autologous hematopoietic recovery by day 52. One patient with ALL died of relapse 150 days after transplant. With a median follow-up of 23 months (range 100 days to 3.6 y), 14 out of 15 patients are alive, and one-year event-free survival and overall survival are 83% and 92% respectively. In conclusion, we show that continuous infusion of MMF is feasible and well-tolerated. In this pilot PK study, we observed no toxic deaths, excellent engraftment, and low rates of grade III-IV aGVHD and cGVHD. The PK studies showed significantly lower half-life and higher drug clearance in pediatric BMT recipients compared to stable pediatric renal transplant patients. This regimen deserves further validation in a larger cohort of pediatric patients undergoing myeloablative transplantation. Disclosures: Off Label Use: Use of Mycophenolate Mofetil for Graft versus Host Disease Prophylaxis.

Chemotherapy-induced mucosal barrier dysfunction

Gut toxicity, or mucositis, is a major dose-limiting side effect of chemotherapy that until recently received very little attention. Despite significant research, the mechanisms that underpin chemotherapy-induced gut toxicity (CIGT) remain unclear. Recently however, there has been renewed interest in the role tight junctions play in the pathogenesis of CIGT and associated diarrhea. Thus, this review will cover the role of tight junctions in maintaining gastrointestinal homeostasis and touch on recently proposed mechanisms of how tight junctions may contribute to the development of chemotherapy-induced diarrhea. There is a wealth of anecdotal evidence regarding the role of tight junctions in the pathogenesis of gut toxicity. However, few studies have quantified or assessed the molecular changes in tight junctions in response to chemotherapy. This review will highlight the major findings of these studies and discuss the potential mechanisms by which tight junction disruption and mucosal barrier dysfunction may contribute to diarrhea. The significant clinical and economic impact associated with CIGT and diarrhea has only recently been appreciated. This has prompted significant research efforts in an attempt to reveal the pathophysiology of this debilitating complication. Renewed interest has been shown regarding the role of tight junctions in not only maintaining gastrointestinal health, but also contributing to mucosal barrier injury and diarrhea development. More detailed research into the effect chemotherapy has on the molecular characteristics of tight junctions will lead to a better understanding of the pathophysiology of CIGT and may uncover the therapeutic potential of tight junctions in treating diarrhea.

Running Interference? Exercise and PCB-Induced Changes in the Gut Microbiome

Trillions of diverse bacteria live in the gastrointestinal tract in a community collectively known as the gut microbiome. Changes in the makeup of the gut microbiome have been implicated in a variety of diseases, including obesity and diabetes,1 both of which also are affected by exercise, diet, and other lifestyle behaviors.2 Likewise, exposure to the industrial pollutants polychlorinated biphenyls (PCBs) has been shown to impair glucose homeostasis in mice,3 suggesting it could increase the risk for diabetes, and obese people have been reported to have higher body burdens of PCBs than lean people.4 A novel study in EHP looks at the interaction among the gut microbiome, exercise, and PCB exposure in mice.5 The study showed that short-term oral exposure to an environmentally relevant mixture of PCBs was associated with reduced abundance and diversity of gut microbes. However, mice that exercised daily before PCB dosing began showed fewer changes to the gut microbiome than sedentary mice. The mice were about a year old, comparable to middle age for people. Half the mice exercised voluntarily on a running wheel, running an average of 10–12 km per day; the other half had locked wheels in their cages. After a five-week exercise period all mice were dosed with a mixture of PCB138, PCB153, and PCB180 for two days. These PCB congeners and the amounts given reflect those found in high concentrations in contaminated fish from the Great Lakes, according to senior author Michal Toborek, Leonard M. Miller Professor of Biochemistry and Molecular Biology at the University of Miami Miller School of Medicine. Fecal samples collected from the mice after the exercise period and again after PCB treatment were analyzed for changes in the gut microbiome.5 The mice that ran weighed 30% less than the sedentary animals after the five-week exercise period. In sedentary mice PCB exposure was associated with a nearly sixfold decrease in Proteobacteria, whereas mice that exercised did not experience a significant drop in Proteobacteria abundance. Compared with sedentary mice, exercised mice showed greater bacterial diversity. They had up to 24 times greater abundance of several Firmicutes species, whereas Tenericutes, Bacteroidetes, and other Firmicutes species were less abundant. The most striking difference between exercised and sedentary mice was a dramatic 360-fold reduction in Erysipelotrichaceae abundance in the former.5 “We didn’t expect to see so many changes in a 48-hour window,” Toborek says. In this study, the mice’s running activity was monitored via a direct data link to a computer running Clocklab™ Analysis software. Mice that exercised before PCB exposure showed fewer changes to the gut microbiome than sedentary mice. The physiological importance of the changes in gut bacteria remains to be determined. Other researchers have reported depletion of Firmicutes and increased levels of Proteobacteria in the guts of patients with inflammatory bowel disease6 and a relative abundance of Erysipelotrichaceae in patients with colorectal cancer, compared with healthy controls.7 Studies suggest that Bacteroidetes species are reduced and Firmicutes are elevated in obesity,8 and that the levels reciprocally reverse after weight loss.9 Toborek plans to expose mice to PCBs for longer times to examine how chronic exposure affects the gut microbial profile. Mice also will be exercised after treatment with PCBs to determine the effects of physical activity over time. Other experiments will look at the influence of the gut microbiome on the chemical fate of PCBs. Gut microbes produce enzymes that chemically alter drugs, hormones, and carcinogens. These biotransformations can either diminish or enhance the toxicity of substances such as PCBs.10,11 “It’s well known that diet may alter biological responses to environmental [toxicants], but the role of gut flora in toxification processes has rarely been evaluated,” says Larry W. Robertson, a University of Iowa professor of occupational and environmental health. Toborek’s results give new insights into the relationship between the gut microbiome and toxicity as well as the ways in which exercise alters the gut microbiome and toxicity. In addition to promoting diet and exercise for good health, Robertson says, perhaps we should include the role our gut flora play in wellness.

Editorial introductions

Editorial introductions

Diagnosis and Management of Clostridium difficile Infection

Clostridium difficile infection (CDI) is increasing in frequency and severity in and out of the hospital, with a high probability of recurrence after treatment. The recent literature on CDI was reviewed using PubMed to include recent publications dealing with diagnosis and therapy. Real-time polymerase chain reaction is a sensitive and useful diagnostic test for CDI but there are growing concerns of false-positive test results if the rate of CDI is low in the patient population providing samples and/or if the population being studied commonly includes people with C difficile colonization. Recommended therapy of CDI includes oral metronidazole for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases, each given for 10 days. Colectomy is being performed more frequently in patients with fulminant CDI. For treatment of first recurrences the drug used in the first bout can be used again and for second recurrences longer courses of vancomycin often are given in a tapered dose or intermittently to allow gut flora reconstitution, or other treatments including fidaxomicin may be used. Bacteriotherapy with fecal transplantation is playing an increasing role in therapy of recurrent cases. Metagenomic studies of patients with CDI during successful therapy are needed to determine how best to protect the flora from assaults from antibacterial drugs and to develop optimal therapeutic approaches. Immunotherapy and immunoprophylaxis offer opportunities to prevent CDI, to speed up recovery from CDI, and to eliminate recurrent infection. Humanized monoclonal antitoxin antibodies and active immunization with vaccines against C difficile or its toxins are both in development and appear to be of potential value.

Modulation of protein fermentation does not affect fecal water toxicity: a randomized cross-over study in healthy subjects.

ObjectiveProtein fermentation results in production of metabolites such as ammonia, amines and indolic, phenolic and sulfur-containing compounds. In vitro studies suggest that these metabolites might be toxic. However, human and animal studies do not consistently support these findings. We modified protein fermentation in healthy subjects to assess the effects on colonic metabolism and parameters of gut health, and to identify metabolites associated with toxicity.DesignAfter a 2-week run-in period with normal protein intake (NP), 20 healthy subjects followed an isocaloric high protein (HP) and low protein (LP) diet for 2 weeks in a cross-over design. Protein fermentation was estimated from urinary p-cresol excretion. Fecal metabolite profiles were analyzed using GC-MS and compared using cluster analysis. DGGE was used to analyze microbiota composition. Fecal water genotoxicity and cytotoxicity were determined using the Comet assay and the WST-1-assay, respectively, and were related to the metabolite profiles.ResultsDietary protein intake was significantly higher during the HP diet compared to the NP and LP diet. Urinary p-cresol excretion correlated positively with protein intake. Fecal water cytotoxicity correlated negatively with protein fermentation, while fecal water genotoxicity was not correlated with protein fermentation. Heptanal, 3-methyl-2-butanone, dimethyl disulfide and 2-propenyl ester of acetic acid are associated with genotoxicity and indole, 1-octanol, heptanal, 2,4-dithiapentane, allyl-isothiocyanate, 1-methyl-4-(1-methylethenyl)-benzene, propionic acid, octanoic acid, nonanoic acid and decanoic acid with cytotoxicity.ConclusionThis study does not support a role of protein fermentation in gut toxicity. The identified metabolites can provide new insight into colonic health.Trial RegistrationClinicalTrial.gov NCT01280513

Chemotherapy-induced gut toxicity: are alterations to intestinal tight junctions pivotal?

Chemotherapy-induced gut toxicity (CIGT) is a frequent, debilitating and dose-limiting side effect of anti-cancer cytotoxic therapies. Despite much research, many of the underlying mechanisms remain poorly understood. Recently, there has been renewed interest in the role that intestinal permeability and tight junctions play in the pathogenesis of chemotherapy-induced gut toxicity. Tight junctions have been linked with many of the known hall marks of toxicity including pro-inflammatory cytokines and pathogenic bacteria. In this critical review, we highlight the research literature addressing modifications in tight junctions following chemotherapy administration and how tight junctions may be implicated in the pathophysiology of CIGT.

Targeting the Notch Pathway: Twists and Turns on the Road to Rational Therapeutics

The road leading to new therapeutics is often bumpy and occasionally treacherous. Attempts to target Notch in cancer exemplify the challenges associated with such ventures. Notch is an increasingly attractive cancer target: It has direct oncogenic roles in multiple hematologic malignancies (as reviewed); it is essential for tumor angiogenesis; it has important roles in innate and adaptive immune cell function; and it is needed to establish and maintain certain stemcell populations, suggesting a role in maintenance of tumor-initiating cells. However, Notch is also tumor suppressive in squamous epithelia and may have antioncogenic effects in blood vessels and myeloid cells as well. In addition, Notch inhibitors have problematic on-target gut toxicity. These concerns are mitigated by the results of Tolcher et al reported in Journal of Clinical Oncology. Using several dosing schedules, Tolcher et al show that RO4929097, a -secretase inhibitor (GSI), has a tolerable safety profile and a hint of antitumor activity in patients with refractory metastatic or locally advanced solid tumors. Pharmacodynamic studies have shown modest but significant evidence of Notch inhibition. These findings are in line with prior experience and constitute a modest step forward toward realizing Notch-directed therapies in cancer. Here, we review the Notch pathway and some of the challenges and opportunities it presents to clinical investigators.

Reversal of glucocorticoid resistance by the g-secretase inhibitor PF-03084014 in T-cell acute lymphoblastic leukemia.

9553 Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer frequently characterized by activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of g-secretase inhibitors (GSIs), but subsequent work found that inhibition of NOTCH signaling is characterized by a lack of cytotoxicity of human T-ALL cells and severe gastrointestinal toxicity. Our preliminary results show that combination therapy of generic GSIs plus glucocorticoids has improved antileukemic effects in vitro and in vivo and reduced gut toxicity. Here we characterized the interaction between PF-03084014, a clinically relevant GSI, and dexamethasone in preclinical models of glucocorticoid resistant T-ALL. Methods: Studies were performed using a panel of human T-ALL cell lines and primary patient samples. The effect of GSI treatment plus glucocorticoids was assessed using cell viability, cell cycle, and proliferation assays....

Abstract 4358: Monoclonal antibodies to Notch receptors may enable targeting of tumor autonomous and tumor micro-environmental processes

Abstract AVEO has developed a series of inducible mouse models of cancer which, through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding is consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit various Notch receptors. Characterization of monoclonal antibodies targeting Notch1 or Notch3 through cell-based and biochemical studies demonstrated that these antibodies bound with high affinity and high specificity to the ligand binding domains of the Notch receptors, prevented ligand mediated activation of the target receptor, and specifically repressed Notch-dependent signaling with high potency. Effective inhibition of functional angiogenesis was observed upon anti-Notch1 antibody treatment in both in vitro and in vivo models. Significantly, specific inhibition of Notch1 by this antibody did not result in the dose-limiting gut toxicity observed with pan-Notch inhibitors such as gamma-secretase inhibitors. Humanized versions of the Notch1 monoclonal antibodies have been generated and characterized. Inhibition of tumor growth by the Notch3 monoclonal antibody was effected through tumor cell autonomous mechanisms. To identify tumors that are dependent upon tumor autonomous Notch signaling, gene expression profiles were correlated with Notch pathway dependence in human cancer cell lines. Expression of specific downstream targets was highly correlated with sensitivity of human cancer cell lines to inhibition of ligand-dependent Notch signal. This biomarker of Notch pathway dependence successfully predicted that a subset of Kras mutant pancreatic and colon cancer cell lines would be highly sensitive to Notch pathway inhibition. Moreover, identification of Notch1 and Notch3-specific target genes further enables selection of tumors that will respond to monoclonal antibodies specifically targeting one or other, or both of these receptors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2011-4358

Nutrition Modulation of Gastrointestinal Toxicity Related to Cancer Chemotherapy

Chemotherapy-induced gut toxicity is a major dose-limiting toxicity for many anticancer drugs. Gastrointestinal (GI) complications compromise the efficacy of chemotherapy, promote overall malnutrition, aggravate cancer cachexia, and may contribute to worsened prognosis. The GI tract is an attractive target for nutrition modulation, owing to its direct exposure to the diet, participation in uptake and metabolism of nutrients, high rate of cell turnover, and plasticity to nutrition stimuli. Glutamine, ω-3 polyunsaturated fatty acids, and probiotics/prebiotics are therapeutic factors that potentially modulate GI toxicity related to cancer treatments. Preclinical and clinical evidence are reviewed to critically define plausible benefits of these factors and their potential development into adjuncts to cancer chemotherapy. Mechanisms underlying the action of these nutrients are being unraveled in the laboratory. Optimal strategies to translate these findings into clinical care still remain to be elucidated. Key questions that remain to be answered include the following: which nutrient or combination of nutrients is selected for which patient and chemotherapy regimen? What mechanisms are responsible for modulation, and how are nutrient(s) administered in a clinically optimal manner? Research exploring interactions between different nutrients in GI protection is ongoing and demands further understanding. How nutrition preparations given to chemotherapy-treated patients are formulated in terms of component selection and dose optimization should be carefully studied and justified.

510 5-azacitidine restores and amplifies the response to bicalutamide and radiotherapy on preclinical models of androgen receptor expressing or deficient prostate tumors

Pre-clinical data have shown that PARP inhibitors (PARPi) may increase the efficacy of radiotherapy in prostate cancer. However, it is uncertain as to whether PARPi lead to clonogenic kill when combined with radiotherapy (RT).We tested the PARP inhibitor AZD-2281 as a radiosensitizing agent under oxic and hypoxic conditions for clonogenic survival in vitro and in vivo using the human prostate cancer cell line, 22Rv1. In addition, the effects of PARPi + RT on normal tissue were investigated using a crypt clonogenic assay.AZD-2281 inhibited cellular PARP activity under both oxic and hypoxic conditions. The addition of AZD-2281 radiosensitized 22Rv1 cells under oxia, acute hypoxia and chronic hypoxia in vitro. The combination of AZD-2281 with fractionated radiotherapy resulted in a significant growth delay and clonogenic kill in vivo. No increased gut toxicity was observed using this combined PARPi + radiotherapy regimen.This is the first preclinical study to demonstrate direct clonogenic kill in vivo by the addition of AZD-2281 to radiotherapy. As we did not observe gut toxicity, the use of PARPi in the context of prostate cancer radiotherapy warrants further investigation in clinical trials.

Abstract 5170: Monoclonal antibodies to Notch receptors inhibit tumor maintenance

Abstract AVEO has developed a series of inducible mouse models of cancer which, through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding is consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit various Notch receptors. Characterization of a Notch1-specific monoclonal antibody through cell-based and biochemical studies demonstrated that the antibody bound to the Notch1 ligand binding domain with high affinity, prevented ligand mediated activation of the receptor, and specifically repressed Notch1-dependent signaling with high potency. Mice treated with the Notch1 antibody exhibited altered T cell fate specification and loss of hair pigmentation, as expected for loss of Notch1 function. Significantly, specific inhibition of Notch1 by this antibody did not result in the dose-limiting gut toxicity observed with pan-Notch inhibitors such as gamma-secretase inhibitors. However, strong inhibition of functional angiogenesis was observed upon antibody treatment in both in vitro and in vivo models. The apparent lack of toxicity of this antibody in mouse models suggests that inhibition of Notch1 could be effectively combined with other therapies to enhance anti-angiogenic effects, or to overcome resistance to VEGF/VEGFR inhibition. To identify tumors that are dependent upon tumor autonomous Notch signaling the expression of Notch pathway related genes was correlated with Notch pathway dependence in human cancer cell lines. Active Notch signaling alone did not predict dependence upon Notch, but expression of a single Notch target gene, HeyL, was highly correlated with sensitivity of human cancer cell lines to inhibition of ligand-dependent Notch signal. The utility of this biomarker was further confirmed by the identification of a subset of Kras mutant pancreatic and colon cancer cell lines that were subsequently demonstrated to be highly sensitive to Notch pathway inhibition. Hence, HeyL expression may serve as a predictive biomarker of Notch-dependent tumors. These data support the clinical development of AVEO's humanized Notch antibodies for the treatment of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5170.

Falling Serum Albumin Predicts Severity of Acute Graft-Vs.-Host Disease and Non-Relapse Mortality After Non-Myeloablative Allogeneic Hematopoietic Cell Transplantation.

Abstract 1146Poster Board I-168 BACKGROUNDAcute graft-vs.-host disease (aGVHD) varies in its intensity and response to therapy. Biomarkers capable of differentiating treatment-responsive from severe aGVHD at symptom onset may enable risk-stratified therapy. With the advent of prophylactic ursodiol, liver aGVHD has become rare, and gut involvement is responsible for much of the morbidity and mortality associated with severe aGVHD. Protein loss through altered epithelial tight junctions, and ultimately through mucosal necrosis, results in hypoalbuminemia in severe aGVHD (Weisdorf et al., Gastroenterology 1983 85:1076-81). We hypothesized that intestinal protein loss correlated with severity of aGVHD and thus predicted outcome. To test this hypothesis, we retrospectively evaluated changes in serum albumin as a marker of protein loss at the onset of aGVHD symptoms. To reduce the confounding effect of regimen-related gut toxicity, we restricted our analysis to patients who received non-myeloablative conditioning. PATIENTS AND METHODSAmong 708 recipients of a first non-myeloablative allogeneic HCT at our Center, 412 (58%) developed aGVHD grade II–IV. We retrospectively evaluated these 412 patients (336 grade II; 76 grade III/IV). Patients were conditioned with 2 Gy total body irradiation, with or without fludarabine 90 mg/m2. Post-grafting immunosuppression consisted of cyclosporine and mycophenolate mofetil, and corticosteroids were used as first-line treatment of aGVHD. Median patient age was 55 (range, 1–75) years. 170 patients (41%) had related donors. Among the 242 patients (59%) with unrelated donors, there were 31 HLA-allele-mismatched donors, 28 HLA-antigen-mismatched donors, and 6 allele and antigen mismatched donors. We reviewed all serum albumin values from 30 days before to 45 days after clinical onset of aGVHD. The lowest albumin value in the 30 days before HCT was considered a patient's baseline value. Trends in serum albumin before and after aGVHD onset were compared among patients with peak grade II vs. peak grade III/IV aGVHD. We also compared 6-month estimates of non-relapse mortality (NRM) associated with changes in serum albumin and the performance of falling serum albumin as a predictor of grade III/IV aGVHD. RESULTSClinical onset of aGVHD in the study cohort occurred at a median of 35 days after HCT. Patients who went on to develop severe (grade III/IV) aGVHD showed sharp decreases from baseline serum albumin levels; these decreases were already apparent at the onset of clinical aGVHD (Fig. 1). In contrast, patients whose aGVHD never exceeded grade II showed no changes on average from baseline serum albumins at aGVHD onset. Receiver-operator characteristic (ROC) curves demonstrated that among patients with new-onset symptoms of aGVHD requiring treatment, a drop from baseline in serum albumin of 0.5 g/dL optimally discriminated between those who went on to develop severe aGVHD versus those whose aGVHD peaked at grade II (ROC area under curve, 0.75). A drop from baseline albumin of 0.5 g/dL at onset of aGVHD symptoms was also associated with an increased risk of NRM at 6 months after aGVHD onset (28% vs. 8%, HR 2.13 [1.3–3.4], p=0.002; Fig. 2). CONCLUSIONA sharp decrease in serum albumin may precede and predict severe aGVHD as well as NRM. Serial measurement of serum albumin is an inexpensive and readily available potential prognostic biomarker in patients with new-onset symptoms of aGVHD after non-myeloablative allogeneic HCT. In combination with other biomarkers or clinical predictors of aGVHD severity, the kinetics of serum albumin may enable specific and risk-stratified targeting of therapies for aGVHD, reserving intensive and protracted immunosuppression for the small percentage of patients at highest risk of severe aGVHD.FIGURES: [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparitive study of efficacy, tolerability of four field box technique vs. two field anterior posterior technique in locally advanced carcinoma cervix:A prospective analysis

Purpose: The pelvic failure rate decreases with increase in the radiation dose but the complications increase. The four field pelvic technique has a theoretical advantage of providing a higher tumor dose with less dose to the surrounding normal tissue thus reducing the complications.Result: Eighty-three patients completed treatment, 42 in Arm A and 41 in Arm B. The complete response achieved in the two groups was 85.75% and 87.8% (p = 0.67). Skin reactions were more in Arm B (p < 0.05). Grade II GIT symptoms were more common in both the groups (p = 0.75). Grade I GUT toxicity was the most common toxicity in both the arms (p = 0.38). The most common hematological toxicity in the two arms was of grade II (p = 0.78).Material and method: After satisfying the eligibility criteria histo-pathologically proven locally advanced carcinoma cancer cervix patients were randomized to four (Arm A) or two (Arm B) field techniques.Conclusions: Both two and four field box techniques are equally effective and feasible as stastically insignificant difference in the response rate and acute toxicities was observed in the two arms.

NOTCH inhibition and glucocorticoid therapy in T-cell acute lymphoblastic leukemia

Inhibition of NOTCH1 signaling with gamma-secretase inhibitors (GSIs) has been proposed as a molecularly targeted therapy in T-cell acute lymphoblastic leukemia (T-ALL). However, GSIs seem to have limited antileukemic activity in human T-ALL and are associated with severe gastrointestinal toxicity resulting from inhibition of NOTCH signaling in the gut. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid sensitivity and co-treatment with glucocorticoids inhibited GSI-induced gut toxicity. Thus, combination therapies with GSIs plus glucocorticoids may offer a new opportunity for the use of anti-NOTCH1 therapies in human T-ALL.

γ-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia

SummaryGamma-secretase inhibitors (GSIs) block the activation of oncogenic NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor auto-up-regulation and induced apoptotic cell death through induction of BIM expression. GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of Klf4, a negative regulator of cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of Ccnd2 and protected mice from developing intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

Inhibition of NOTCH1 Signaling and Glucocorticoid Therapy in T-ALL

Activating mutations in NOTCH1 are present in over 50% of T-cell acute lymphoblastic leukemias (T-ALL). Consequently, inhibition of NOTCH1 signaling with small molecule γ-secretase inhibitors (GSIs) has been proposed as a targeted therapy for this disease. However, GSIs fail to induce robust apoptosis in T-ALL cells and their clinical application has been limited by the development of severe gastrointestinal toxicity (mucous diarrhea and goblet cell metaplasia) resulting from systemic inhibition of NOTCH signaling. Here we show that combination therapy with GSIs plus glucocorticoids can reverse both glucocorticoid resistance in T-ALL and the occurrence of GSI-induced gut toxicity in vivo. The development of glucocorticoid resistance in T-ALL has been associated with the loss of a positive auto-regulatory transcriptional feedback loop required for glucocorticoid induced apoptosis. Using gene expression profiling and ChIP-on-chip analysis we demonstrated that HES1, a transcriptional repressor controlled by NOTCH1, inhibits specific regulatory elements in the glucocorticoid receptor promoter required for effective glucocorticoid receptor autoupregulation and glucocorticoid induced cell death. Consequently, downregulation of HES1 via inhibition of NOTCH1 signaling with GSIs or HES1 inactivation via shRNA knockdown restores glucocorticoid receptor autoupregulation and reverses glucocorticoid resistance in T-ALL cell lines and primary patient samples. Consistent with these results, combination therapy with dexamethasone and GSIs was highly effective in a mouse xenograft model of glucocorticoid resistant T-ALL. Surprisingly, these studies demonstrated not only improved antileukemic responses in mice treated with dexamethasone plus a GSI compared with dexamethasone or placebo treated controls (P<0.05), but also decreased mortality associated with GSI-induced gut toxicity in this model (P<0.01). These results suggested that glucocorticoids may have an enteroprotective effect against GSI-induced gut toxicity. Detailed histological analysis demonstrated a block of proliferation and increased goblet cell numbers in the intestine of GSI treated animals and a moderate increase in proliferation accompanied by increased Paneth cell numbers in the dexamethasone treated group. Notably, cotreatment with dexamethasone plus a GSI completely reversed the induction of goblet cell metaplasia typically induced by systemic inhibition of NOTCH signaling with GSIs. Likewise, dexamethasone also protected RBPJ-K conditional knockout mice, a genetic model of loss of NOTCH signaling, from developing goblet cell metaplasia in the gut. Interestingly, gene expression profiling analysis showed a marked upregulation of cyclin D2 expression in the gut of glucocorticoid treated mice. Moreover, dexamethasone treatment failed to protect Ccnd2 knockout animals from developing GSI-induced metaplasia, demonstrating a critical role of Ccnd2 upregulation in the protective effects of dexamethasone against GSI-induced gut toxicity. Overall, these results uncover a NOTCH1-HES1-glucocorticoid receptor regulatory axis in glucocorticoid resistant T-ALL and demonstrate an unanticipated protective effect of glucocorticoids against GSI-induced gut toxicity. Our studies warrant the clinical testing of glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

Tyrosine kinase inhibitors and gut toxicity: a new era in supportive care

Tyrosine kinase inhibitors and gut toxicity: a new era in supportive care

Inhibition of NOTCH1 Signaling Reverses Glucocorticoid Resistance in T-ALL.

Gamma-secretase inhibitors (GSIs), which block the activation of the NOTCH1 receptor, are currently being tested in the treatment of T-cell lymphoblastic leukemias (T-ALL) with activating mutations in the NOTCH1 gene. However, inhibition of NOTCH1 signaling induces only a delayed cytostatic antileukemic effect with little or no apoptosis and GSI treatment is associated with severe gastrointestinal toxicity, which limits the clinical application of these molecularly targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids reverses glucocorticoid resistance and is highly effective against T-ALL. Inhibition of NOTCH1 signaling with CompE, a highly active GSI, in combination with dexamethasone, induced apoptotic cell death in glucocorticoid-resistant T-ALL cell lines. The synergistic antileukemic effect of GSIs and glucocorticoids was effectively rescued by forced expression of ICN1, an intracellular form of NOTCH1 that does not require gamma-secretase processing; or by treatment with the glucocorticoid receptor antagonist RU486. Reversal of glucocorticoid resistance by CompE treatment was mediated by transcriptional upregulation of BIM, a BH3-only proapototic factor previously involved in glucocorticoid induced cell death. Thus, shRNA knock down of BIM effectively impaired the ability of CompE plus dexamethasone to induce programmed cell death in leukemic lymphoblasts. Using a mouse xenograft model of glucocorticoid-resistant T-ALL we demonstrated that the combination of DBZ, a GSI with well established activity in vivo, and dexamethasone is highly effective against glucocorticoid resistant T-ALL in vivo. All animals treated with DBZ plus dexamethasone showed complete clearance of leukemic lymphoblasts and improved survival, while mice treated with vehicle or dexamethasone alone died because of disease progression (P<0.05). In these studies, treatment with DBZ alone was associated with accelerated mortality because of severe intestinal toxicity characterized by goblet cell metaplasia in the small intestine. Surprisingly, mice treated with DBZ plus dexamethasone were protected clinically and histologically from GSI-induced intestinal toxicity. Pharmacokinetic analysis demonstrated that the interaction of dexamethasone and DBZ in the gut was not mediated by increased DBZ metabolism induced by dexamethasone. Molecular characterization of GSI induced gut toxicity demonstrated transcriptional upregulation of Klf4, a critical regulator of goblet cell differentiation, in the intestines of animals treated with DBZ. Klf4 regulation downstream of NOTCH signaling is mediated by HES1, a transcriptional repressor regulated by NOTCH. Importantly, dexamethasone treatment blocked the upregulation Klf4 induced by GSI treatment in vivo, suggesting that modulation of Klf4 expression may mediate the enteroprotective effects of glucocorticoids against GSI-induced toxicity. Overall, these results support a role for the combination of glucocorticoids plus GSIs in the treatment of glucocorticoid resistant T-ALL.