Regimen For Bone Marrow Transplantation Research Articles

Complications of total-body irradiation in allogeneic bone marrow transplantation: A review article

Hematopoietic stem cell transplantation commonly known as bone marrow transplantation (BMT) or allogeneic BMT (using healthy blood stem cells from a donor) is the preferred therapeutic option for numerous blood-related conditions, both malignant and non-malignant. It is often the sole therapy strategy and essential for relapsed and refractory hematologic malignancies. There have studies regarding BMT on regimen containing total body irradiation (TBI) and a regimen without TBI. It is expected that TBI-based conditioning regimens provide better antitumor effects than chemotherapy regimens. The primary objective of TBI is to eradicate the recipient's bone marrow, facilitating the successful engraftment of donor bone marrow. Acute lymphoid leukemia (ALL) is the principal indication for TBI in bone marrow transplantation. Other diseases including Hodgkin’s lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma (MM), etc., may benefit from TBI-based regimens; however, TBI use is associated with many side effects.The main complications of patients who underwent TBI-containing conditioning regimens in bone marrow transplantation are vomiting and nausea, with frequencies of approximately 66% and 35%, respectively. However, these events are easily managed. Acute complications include stomatitis, diarrhea, loss of appetite, temporary loss of taste, rash and asthenia. Moreover, veno-occlusive disease, interstitial pneumonitis, lung side effects, growth hormone deficiency, neurological side effects, cataracts, renal toxicity, endocrine impairments, and infertility are other complications in patients who underwent TBI-containing conditioning regimens in bone marrow transplantation. In review article, a complication of total-body irradiation in allogeneic bone marrow transplantation was assessed.

Enrollment Lessons from a Biological Assignment Study of Marrow Transplantation versus Standard Care for Adolescents and Young Adults with Sickle Cell Disease: Considerations for Future Gene and Cellular Therapy Trials

We previously conducted a single-arm feasibility study (STRIDE1) of myeloablative bone marrow transplantation (BMT) in adolescents and young adults with sickle cell disease (SCD). The trial identified donors before entry, enrolled well, and found no unexpected regimen-related toxicity. Although many single-arm studies have been published, there are no controlled trials of either BMT or gene therapy in SCD. Therefore, we designed a comparative trial by biological assignment (available donor versus no donor). This multicenter National Institutes of Health-funded study (Blood and Marrow Transplant Clinical Trials Network 1503; STRIDE2) enrolled patients between 2016 and 2021 at 35 sites. Lagging recruitment led to study closure, and here we report the impediments to accrual. The BMT regimen and entry criteria were from STRIDE1, and 2-year survival was the primary endpoint. To minimize selection bias from prior HLA typing, STRIDE2 excluded individuals with previously identified donors. Accrual was stopped at 69% of target (138 enrolled; assigned 28 with donor, 96 with no donor). Barriers to enrollment included lower than expected frequency of HLA-matched related and unrelated donors; loss of enrollees owing to previously identified donors; conventional care arm dissuading some seeking BMT; challenging short-term endpoints in SCD, including incomplete documentation of sickle pain episodes; state Medicaid (primary insurers of SCD) denial of BMT coverage for adult SCD despite the study having secured Coverage with Evidence Development from the Center for Medicare & Medicaid Services; slowed accrual in 2019 to 2021 during the Coronavirus disease 2019 pandemic; and restriction of BMT resourcing for nonmalignant diseases by academic medical (cancer) centers. Social obstacles and access to BMT centers also limited entry, as did practitioner and participant concerns over suitability, cost, and toxicity. Planning for future controlled trials of curative therapy in SCD and other nonmalignant diseases likely will meet these enrollment challenges. Lessons from this trial may aid the development of future comparative studies.

Automatic Segmentation of Target Structures for Total Marrow and Lymphoid Irradiation in Bone Marrow Transplantation.

The use of total marrow and lymphoid irradiation (TMLI) as part of conditioning regimens for bone marrow transplantation is trending due to its advantages in disease control and low toxicity. Accurate contouring of target structures such as bone and lymph nodes plays an important role in irradiation planning. However, this process is often time-consuming and prone to inter-observer variation. Recently, deep learning methods such as convolutional neural networks (CNNs) and vision transformers have achieved tremendous success in medical image segmentation, therefore enabling fast semiautomatic radiotherapy planning. In this paper, we propose a dual-encoder U-shaped model named DE-Net, to automatically segment the target structures for TMLI. To enhance the learned features, the encoder of DE-Net is composed of parallel CNNs and vision transformers, which can model both local and global contexts. The multi-level features from the two branches are progressively fused by intermediate modules, therefore effectively preserving low-level details. Our experiments demonstrate that the proposed method achieves state-of-the-art results and a significant improvement in lymph node segmentation compared with existing methods.

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies

Total marrow irradiation(TMI) has significantly advanced radiation conditioning for hematopoietic cell transplantation inhematologicmalignancies by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. However, the relapse rate remains high, and the lack of a preclinical TMI model has hindered scientific advancements. To accelerate TMI translation to the clinic, we developed a TMI delivery system in preclinical models. A Precision X-RAD SmART irradiator was used for TMI model development. Images acquired with whole-body contrast-enhanced computed tomography (CT) were used to reconstruct and delineate targets and vital organs for each mouse. Multiple beam and CT-guided Monte Carlo-based plans were performed to optimize doses to the targets and to vary doses to the vital organs. Long-term engraftment and reconstitution potential were evaluated by a congenic bone marrow transplantation (BMT) model and serial secondary BMT, respectively. Donor cell engraftment was measured using noninvasive bioluminescence imaging and flow cytometry. Multimodal imaging enabled identification of targets (skeleton and spleen) and vital organs (eg, lungs, gut, liver). In contrast to total body irradiation (TBI), TMI treatment allowed variation of radiation dose exposure to organs relative to the target dose. Dose reduction mirrored that in clinical TMI studies. Similar to TBI, mice treated with different TMI regimens showed full long-term donor engraftment in primary BMT and second serial BMT. The TBI-treated mice showed acute gut damage, which was minimized in mice treated with TMI. A novel multimodal image guided preclinical TMI model is reported here. TMI conditioning maintained long-term engraftment with reconstitution potential and reduced organ damage. Therefore, this TMI model provides a unique opportunity to study the therapeutic benefit of reduced organ damage and BM dose escalation to optimize treatment regimens in BMT and hematologic malignancies.

Radiation causes tissue damage by dysregulating inflammasome-gasdermin D signaling in both host and transplanted cells.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.

Dispersive micro solid phase extraction of busulfan from plasma samples using novel mesoporous sorbent prior to determination by HPLC-MS/MS

Determination of busulfan concentration in patients undergoing bone marrow transplantation is necessary in order to reduce toxic effects and/or graft rejection due to unadjusted dose exposure. A new extraction method namely dispersive micro solid phase extraction (DMSPE) based on mesoporous sorbent was used for cleaning-up the plasma samples. DMSPE coupling with liquid chromatography with tandem mass spectrometry (LC–MS/MS) was implemented for the determination of busulfan dosage in plasma samples. The linear range was found from 10 to 2000 ng/ml. The precision and accuracy were found better than 15% according to Food and drug Administration (FDA) guideline. This method was successfully used to determine the busulfan in patients administrated busulfan as part of the preparative regimen for bone marrow transplantation.

Choice of conditioning regimens for bone marrow transplantation in severe aplastic anemia

AbstractAllogeneic bone marrow transplantation (BMT) is curative therapy for the treatment of patients with severe aplastic anemia (SAA). However, several conditioning regimens can be used for BMT. We evaluated transplant conditioning regimens for BMT in SAA after HLA-matched sibling and unrelated donor BMT. For recipients of HLA-matched sibling donor transplantation (n = 955), fludarabine (Flu)/cyclophosphamide (Cy)/antithymocyte globulin (ATG) or Cy/ATG led to the best survival. The 5-year probabilities of survival with Flu/Cy/ATG, Cy/ATG, Cy ± Flu, and busulfan/Cy were 91%, 91%, 80%, and 84%, respectively (P = .001). For recipients of 8/8 and 7/8 HLA allele-matched unrelated donor transplantation (n = 409), there were no differences in survival between regimens. The 5-year probabilities of survival with Cy/ATG/total body irradiation 200 cGy, Flu/Cy/ATG/total body irradiation 200 cGy, Flu/Cy/ATG, and Cy/ATG were 77%, 80%, 75%, and 72%, respectively (P = .61). Rabbit-derived ATG compared with equine-derived ATG was associated with a lower risk of grade II to IV acute graft-versus-host disease (GVHD) (hazard ratio [HR], 0.39; P < .001) but not chronic GVHD. Independent of conditioning regimen, survival was lower in patients aged >30 years after HLA-matched sibling (HR, 2.74; P < .001) or unrelated donor (HR, 1.98; P = .001) transplantation. These data support Flu/Cy/ATG and Cy/ATG as optimal regimens for HLA-matched sibling BMT. Although survival after an unrelated donor BMT did not differ between regimens, use of rabbit-derived ATG may be preferred because of lower risks of acute GVHD.

Summary of the Third International Workshop on Clinical Tolerance.

The Third International Workshop on Clinical Tolerance was held in Stanford, California, September 8-9, 2017. This is a summary of Workshop presentations of clinical trials designed to withdraw or minimize immunosuppressive (IS) drugs in kidney and liver transplant patients without subsequent evidence of rejection. All clinical protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. Tolerance to HLA matched and mismatched living donor kidney transplants with complete withdrawal of IS drugs without subsequent rejection for up to 14 years of observation was achieved in more than 50 patients enrolled in trials in four medical centers after the establishment of transient or persistent chimerism. Complete IS drug withdrawal without chimerism was reported in a prospective trial of liver transplantation combined with injection of regulatory T cells. IS drug minimization without rejection was reported in recipients of living donor kidney transplants enrolled in the One Study consortium after injection of recipient regulatory T cells, or injection of donor regulatory monocytes or dendritic cells. In conclusion, considerable progress has been made in achieving IS drug withdrawal after cell therapy in recipients of organ transplants.

Bidirectional immune tolerance in nonmyeloablative MHC-mismatched BMT for murine β-thalassemia

AbstractNonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4–driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand–expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and β-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

Classification of Conditioning Regimens for Bone Marrow Transplantation: Historical Background and Current Perspectives

Hematopoietic stem cells transplantation is a current standard treatment for many oncohematological diseases. The milestone of any type of transplantation is the choice of conditioning regimen. This article presents the principles of classification of conditioning regimens in terms of myeloablativity and discusses the concepts of “autologous transplantation”, “high-dose chemotherapy supported by hematopoietic stem cells”, “allogeneic transplantation” and “immunotherapy”. Up-to-date uniform classification of conditioning regimens may serve an important prognostic component in assessing both the risks and efficacy of hematopoietic stem cells transplantation.

Treatment of Pediatric CNS Leukemia With Cranial or Craniospinal Boost in Conjunction With Total Body Irradiation as Part of the Conditioning Regimen for Bone Marrow Transplantation

Treatment of Pediatric CNS Leukemia With Cranial or Craniospinal Boost in Conjunction With Total Body Irradiation as Part of the Conditioning Regimen for Bone Marrow Transplantation

Development and Validation of a High Pressure Liquid Chromatography-UV Method for the Determination of Treosulfan and Its Epoxy Metabolites in Human Plasma and Its Application in Pharmacokinetic Studies.

Treosulfan (l-threitol-1,4-di-methanesulfonate) is a prodrug of a bifunctional alkylating agent that is being used increasingly in pediatric bone marrow transplantation regimens. The activation pathway is a complex reaction, which consists of two consecutive reactions leading to epoxybutane derivatives which are responsible for DNA alkylation. A simple, sensitive high performance liquid chromatography method for the determination of the sum of treosulfan and its epoxy metabolites by UV detection after derivatization with sodium diethyldithiocarbamate in human plasma was developed and validated. Plasma samples containing treosulfan and epoxy metabolites were converted into thiocarbamate derivative with 10% sodium diethyldithiocarbamate. Dinitrobiphenyl was used as an internal standard. The analysis was carried out using a reversed phase C18 column with a mobile phase consisting of methanol-water (65:35, v/v) at a flow rate of 1 mL/min. The eluent was monitored at 254 nm. The standard calibration curve was established between 2.5 and 50 µg/mL, with a correlation coefficient of 0.9987. Intra- and interday precision and accuracy of the method was <8% and met the analytical criteria. Pharmacokinetic parameters were determined in six children who received intravenous treosulfan (dose range 12-24 g/m(2)) in combination with fludarabine prior to blood or marrow transplantation.

Abstract 5495: Identification of markers predictive of phosphoramide mustard (PM) cellular sensitivity in lymphoblast cell lines

Abstract Cyclophosphamide (Cy) a DNA alkylating agent, is commonly used for treatment of several malignancies such as breast cancer and lymphoma. It is also widely used in conditioning regimens for bone marrow transplantation. Cy is a prodrug requiring activation to 4-hydroxycyclophosphamide by hepatic cytochrome P450 enzymes. 4-hydroxy-Cy non-enzymatically converts to active phosphoramide mustard (PM), which produces inter and intra-strand DNA crosslinks causing DNA breaks and hence the cytotoxicity. Although of great clinical utility the dose limiting side effects of Cy as myelosuppression, nephrotoxicity, cardiotoxicity, neurotoxicity and bladder toxicity posses clinical challenge. Genetic polymorphisms in P450 enzymes especially CYP2B6 as well as phase II drug metabolizing enzymes has been shown to partially explain the inter-patient variation in response in some studies however role of pharmacodynamics players is still not clear. Thus, we designed the present study to identify genes predictive of phosphoramide mustard response. We utilized EBV transformed lymphoblast cell lines (LCLs) that are part of International HapMap project as the model system and treated these with PM to by pass the CYP mediated activation of Cy. Since most of the Cy metabolizing genes (CYPs) are expressed in liver but not as much in LCLs, use of PM instead of Cy in the present study enriched our ability to identify genes of relevance to the pharmacodynamic significance. Briefly we treated LCLs with six different concentrations of PM (100, 50, 20, 10, 1, and 0.1 μM), due to stability issues PM was prepared fresh for each experiment. Cell viability was determined 48 hr post drug treatment by MTT assays. Genome-wide gene expression and genotype data was obtained from publically available source and was evaluated for association with PM cellular sensitivity. Top 100 SNPs (p&amp;lt; 2×10-5) mapped to 10 genes of biological significance including CREBBP, DPP10, HYAL3, XRCC1, SNAPC3, CCDC21, PSIP1, RASSF1, and SALL4 CREBBP (CREB binding protein) is a major transcriptional regulator for both estrogen dependent breast cancer signaling and Lymphotoxin b receptor signaling pathways. CREBBP has an intrinsic histone acetyltransferase activity that acts as a scaffold to stabilize additional protein interactions with the transcription complex. Gene-expression association analysis identified 34 genes significantly associated with PM cytotoxicity including EIF4E2, MMP16, MUC6, NDUFA2, NDUFA9 and SDHB. Identification of SNPs in XRCC1 further confirmed the impact of altered DNA damage repair/response pathway in compromising PM response. Our results identified genetic markers of pharmacodynamic significance to Cy response thus opening up opportunities for clinical validation of these in patients receiving Cy and moving a step closer to personalized medicine. Citation Format: Lata Chauhan, Brooke L. Fridley, Alice Wang, Jatinder Kaur Lamba. Identification of markers predictive of phosphoramide mustard (PM) cellular sensitivity in lymphoblast cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2015-5495

Apoptosis-induced activation of HIV-1 in latently infected cell lines.

BackgroundDespite much work, safe and effective approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1 remain an elusive goal. Patients infected with HIV-1 treated with cytotoxic agents or bone marrow transplantation can experience decreases in the reservoir of HIV-1 latently infected cells. Other viruses capable of long-term latency, such as herpesviruses, can sense host cell apoptosis and respond by initiating replication. These observations suggest that other viruses capable of long-term latency, like HIV-1, might also sense when its host cell is about to undergo apoptosis and respond by initiating replication.ResultsPro-monocytic (U1) and lymphoid (ACH-2) HIV-1 persistently infected cell lines were treated with cytotoxic drugs – doxorubicin, etoposide, fludarabine phosphate, or vincristine – and activation of latent HIV-1 was evaluated using assays for HIV-1 RNA and p24 production. Both cell lines showed dose-dependent increases in apoptosis and associated HIV-1 activation following exposure to the cytotoxic agents. Pretreatment of the cells with the pan-caspase inhibitor Z-VAD-FMK prior to exposure to the cytotoxic agents inhibited apoptosis and viral activation. Direct exposure of the latently infected cell lines to activated caspases also induced viral replication. HIV-1 virions produced in association with host cell apoptosis were infectious.ConclusionsThe results indicate that latent HIV-1 can sense when its host cell is undergoing apoptosis and responds by completing its replication cycle. The results may help explain why patients treated with cytotoxic regimens for bone marrow transplantation showed reductions in the reservoir of latently infected cells. The results also suggest that the mechanisms that HIV-1 uses to sense and respond to host cell apoptosis signals may represent helpful new targets for approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1.

Radiation Sensitization of Leukemic Cells for Low Dose Total Body Irradiation

The treatment of pediatric leukemias has been developed through sequential clinical trials designed to improve patient survival and preserve quality of life (Brochstein et al., 1987). A patient's clinical and biological features are predictive of risk of relapse and determine the aggressiveness of the prescribed clinical treatment protocol. Patients determined to be at high risk for recurrence undergo chemotherapy and total body irradiation (TBI) in the preparative regimen for bone marrow transplantation.

Preventive measures for cyclophosphamide-related hemorrhagic cystitis in blood and bone marrow transplantation: an Italian multicenter retrospective study.

Hemorrhagic cystitis (HC) is a troublesome and potentially life-threatening complication of bone marrow transplantation (BMT). HC can appear within a few hours after chemotherapy or after weeks or months. Early-onset HC (EOHC) is usually associated with the conditioning regimen. The main aim of this study was to describe the incidence of EOHC in patients undergoing BMT regimens including high-dose cyclophosphamide (CY) and the effects of the main preventive measures adopted in Italian nursing practice. The authors retrospectively analyzed the clinical records of 158 Italian patients who underwent BMT from 2006-2008. Thirty-one patients (19.6%) developed EOHC. One hundred and forty-seven patients (93%) given high-dose CY were treated with hyperhydration combined with 2-mercaptoethane sulphonate (mesna) and diuresis alkalinization, and only 51 (32.3%) patients were preventively catheterized and received continuous bladder irrigation (CBI). By univariate analysis, prophylactic urethral catheterization and CBI did not decrease EOHC incidence questioning if these measures were to be routinely recommended. Previous studies showed increased discomfort and urinary tract infection in catheterized patients; therefore, nurses may fulfill an important role in balancing the benefits and harms of preventive catheterization and CBI in patients who received BMT conditioning including high-dose CY.

Effect of body mass in children with hematologic malignancies undergoing allogeneic bone marrow transplantation

AbstractThe rising incidence of pediatric obesity may significantly affect bone marrow transplantation (BMT) outcomes. We analyzed outcomes in 3687 children worldwide who received cyclophosphamide-based BMT regimens for leukemias between 1990 and 2007. Recipients were classified according to age-adjusted body mass index (BMI) percentiles as underweight (UW), at risk of UW (RUW), normal, overweight (OW), or obese (OB). Median age and race were similar in all groups. Sixty-one percent of OB children were from the United States/Canada. Three-year relapse-free and overall survival ranged from 48% to 52% (P = .54) and 55% to 58% (P = .81) across BMI groups. Three-year leukemia relapses were 33%, 33%, 29%, 25%, and 21% in the UW, RUW, normal, OW, and OB groups, respectively (P < .001). Corresponding cumulative incidences for transplant-related mortality (TRM) were 18%, 19%, 21%, 22%, and 28% (P < .01). Multivariate analysis demonstrated a decreased risk of relapse compared with normal BMI (relative risk [RR] = 0.73; P < .01) and a trend toward higher TRM (RR = 1.28; P = .014). BMI in children is not significantly associated with different survival after BMT for hematologic malignancies. Obese children experience less relapse posttransplant compared with children with normal BMI; however, this benefit is offset by excess in TRM.

16S rRNA Gene Pyrosequencing Reveals Shift in Patient Faecal Microbiota During High-Dose Chemotherapy as Conditioning Regimen for Bone Marrow Transplantation

Gastrointestinal disturbances are a side-effect frequently associated with haematological malignancies due to the intensive cytotoxic treatment given in connection with bone marrow transplantation (BMT). However, intestinal microbiota changes during chemotherapy remain poorly described, probably due to the use of culture-based and low-resolution molecular methods in previous studies. The objective of our study was to apply a next generation DNA sequencing technology to analyse chemotherapy-induced changes in faecal microbiota. We included eight patients with non-Hodgkin's lymphoma undergoing one course of BMT conditioning chemotherapy. We collected a prechemotherapy faecal sample, the day before chemotherapy was initiated, and a postchemotherapy sample, collected 1 week after the initiation of chemotherapy. Total DNA was extracted from faecal samples, denaturing high-performance liquid chromatography based on amplification of the V6 to V8 region of the 16S ribosomal RNA (rRNA) gene, and 454-pyrosequencing of the 16 S rRNA gene, using PCR primers targeting the V5 and V6 hypervariable 16S rRNA gene regions were performed. Raw sequence data were screened, trimmed, and filtered using the QIIME pipeline. We observed a steep reduction in alpha diversity and significant differences in the composition of the intestinal microbiota in response to chemotherapy. Chemotherapy was associated with a drastic drop in Faecalibacterium and accompanied by an increase of Escherichia. The chemotherapy-induced shift in the intestinal microbiota could induce severe side effects in immunocompromised cancer patients. Our study is a first step in identifying patients at risk for gastrointestinal disturbances and to promote strategies to prevent this drastic shift in intestinal microbiota.

Abstract 424: Depletion of serum levels of miRNA-150 indicates bone marrow damage following total body irradiation.

Abstract Total body irradiation (TBI) is part of the preparative regimen for bone marrow transplantation in lymphoma and leukemia patients. Depending on the aggressiveness of the disease, patients receive a dose between 2 -12 Gray, in 2 Gray fractions over three days. Fast and accurate estimation of the absorbed dose is critical for evaluation of the depletion of cancer cells prior to transplantation. Physical dosimeters and the available biologics have limitations to accurately evaluate the effect of radiation because the normal tissue toxicity as well as the radiation response in cancer cells varies depending on individual's genetics, immune status and other confounding factors. We sought to identify and validate robust and fast responding biomarkers of radiation response, which are repeatedly assayable from body fluids. We have utilized an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the relative abundance of over 600 miRNAs in body fluids. Our studies have identified several evolutionarily and functionally conserved miRNAs in serum and urine collected from two different strains of mice. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed the accurate measurement of changes in serum and urine miRNAs after TBI. miRNAs with good dose response were further investigated for their potential for development as radiation biodosimeters. Hierarchical clustering followed by comparison of changes in individual miRNAs with varying dose (1 - 12 Gray) and time points (1 - 7 days) has identified miRNA-150 as a very promising biomarker for radiation biodosimetry. miRNA-150 is expressed at high level in mature lymphocytes and the abundance of circulating miRNA-150 seems correlating with that of lymphocyte counts. With acute single doses of 1, 2, 4, 6 and 8 Gray, a 30%, 38%, 48% 70% and 72% statistically significant reduction in serum miRNA-150 was observed at 24 hrs, which was further reduced at later time points. Consistent with this data, a time and dose dependent reduction of miRNA-150 was observed after fractionation, with a near complete depletion by 24 hrs after completion of 12 Gray. The kinetics of depletion of serum miRNA-150 following radiation indicate its potential as an alternative to complete blood count (CBC) to evaluate lymphocyte depletion, and hence evaluating damage to the hematopoietic system. The effect of relevant acute single doses and fractionated radiation treatment at various time points will be presented. The utilization of these readouts for understanding the effects and mechanism of action of radioprotectors will also be discussed. Citation Format: Naduparambil K. Jacob, James V. Cooley, Arnab Chakravarti. Depletion of serum levels of miRNA-150 indicates bone marrow damage following total body irradiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 424. doi:10.1158/1538-7445.AM2013-424

Identification of sensitive serum microRNA biomarkers for radiation biodosimetry.

Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual’s response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after therapeutic radiation.