Time Of Bone Marrow Transplantation Research Articles

Stem Cell-Derived Gene Expression Scores Predict Survival and Leukemic Transformation in Myelofibrosis

Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) with survival outcomes ranging from months to years and variable risk of transformation to acute myeloid leukemia. Allogeneic bone marrow transplantation (BMT) can be curative but is associated with high treatment-related morbidity and mortality, therefore accurate risk stratification is important to guide clinical decision making in MF. Current risk prediction models use clinical and/or genomic features but do not consider the properties of the disease-driving stem cell population. Here, we use transcriptomic variation corresponding to both intra- and inter-patient heterogeneity among MF stem cells to generate novel gene expression-based scores predictive of survival and leukemic transformation in MF. To train and validate novel prognostic scores in MF, we identified 358 patients from an MPN registry at the Princess Margaret Cancer Centre (ClinicalTrials.gov Identifier: NCT02760238) from whom peripheral blood (PB) cells were collected near the date of MF diagnosis. All patients were diagnosed with either primary, post-PV, post-ET or pre-fibrotic MF with clinical follow-up of up to 12.2 years. RNA was extracted from unsorted PB mononuclear cells and RNA sequencing (RNAseq) was performed at an average depth of 50 million reads per sample. We randomly split our MF cohort into training (70%; n=250) and test sets (30%; n=108) and utilized a repeated nested cross validation approach together with statistical regression, to generate and assess the performance of models to predict survival within the training set. We tested 36,000 models derived from 36 initial MF-related genesets, ranging from stem-cell specific genesets to the whole transcriptome. The most accurate models by cross validation (median multivariable p-value = 6e-5) were produced from our retrospective identification of highly variable genes in single-cell RNAseq data derived from 82,255 Lin-CD34+ MF stem and progenitor cells across 15 patients (Psaila et al., 2020). Thus, features of intra- and inter-patient heterogeneity among MF stem and progenitor cells proved to be the most relevant for predicting survival. From these features, we derived our final model calculated as the weighted sum of gene expression across 24 genes (termed MPN24). We categorized patients with MPN24 scores above or below the training cohort median as MPN24 high or low, respectively. This model was validated in the test set, with high and low score patients experiencing 5-year survival rates of 71% [95% CI 57-88%] and 21% [95% CI 9%-52%], respectively, when censored at time of BMT (HR = 5.3 [95% CI 2.6-10.5]; p=2.1e-6) (Figure 1A). MPN24 retained independent prognostic value in multivariable analysis incorporating age, sex, DIPSS category, ECOG status, fibrosis grade, constitutional symptoms, and PB blast percentage (adjusted HR = 5.7 [95% CI 2.2-14]; p = 3e-4). Importantly, DIPSS classification remained a significant covariate, indicating that MPN24 and DIPSS capture distinct features of disease. We therefore developed a new three-tier classification scheme integrating both DIPSS and MPN24 scores (Figure 1B). Patients classified as low-, intermediate- or high-risk in this new classification scheme experienced 5-year survival rates of 88.2% [95% CI 77.9% - 99.9%], 39.3% [95% CI 19.9% - 77.7%] and 10.8% [95% CI 2.1% - 55.8%], respectively (likelihood ratio test p = 1e-8). Finally, from the MPN24 genes we derived a 13 gene subscore (MPN13) predictive of leukemic transformation in the training set. Patients in the test set scoring above the 80th percentile from the training set were classified as high risk and the remaining as low risk. Although total leukemic samples were limiting, MPN13 was significantly associated with risk of transformation (p=4.7e-03) with low and high-risk patients experiencing 3-year cumulative incidences of transformation of 5.2% [95% CI 0.2%-10.2%] and 28.6% [95% CI 3.1%-54.0%] respectively, after adjusting for death as a competing risk. Future studies will transfer our scores to the Nanostring platform for testing in independent cohorts. In summary, we used transcriptional variation among MF stem and progenitor cells to derive novel gene expression scores predictive of survival and leukemic transformation and developed a new integrated 3-tier model for predicting risk in MF patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P536: VALUE OF MEASURABLE RESIDUAL DISEASE BY MULTIPARAMETRIC FLOW CYTOMETRY IN NON-PROMYELOCYTIC ACUTE MYELOID LEUKEMIA. REAL WORLD EVIDENCE

Background: Measurable residual disease (MRD) allows recognition of a group of patients with acute myeloid leukemia (AML) with a higher risk of relapse. There are, however, remaining open questions, since clinical studies applied variable techniques, cutoff values and time points. Multiparameter flow cytometry (MFC) is the most accessible method in Argentina, since real time PCR assessment is not available for the majority of the institutions. We designed, therefore, this real-life study using decentralized assessment of MRD by MFC to solve the role of positive MRD at different times during treatment with standard chemotherapy. Aims: Our aim was to assess the impact of positive MRD at the end of induction and after first consolidation (C1) as a prognostic factor for relapse-free survival (RFS) and overall survival (OS) in patients with non-promyelocytic AML. Methods: The registry of the Argentinian Group of Acute Leukemia (GALA) from the Argentine Society of Hematology (SAH) includes data from 20 centers. We retrospectively selected an analytical cohort with all patients with AML, treated with an intensive regimen and in complete remission (CR) after induction, from January 2010 to March 2021. The OS and RFS were estimated with Kaplan-Meier method and its comparison was evaluated by log-rank, censoring patients at the time of bone marrow transplantation (BMT). The cut-off point for MRD analysis was 0.1% evaluated at the end of induction and after C1. A subanalysis of patients with unknown and intermediate cytogenetic risk (CRM modified according to available molecular data of FLT3, NPM1 and CEBPA) was performed. Results: Of the 1,160 patients enrolled in the registry, 493 met the inclusion criteria and 20 were excluded due to lack of MRD data at the end of induction. Among the 473 patients analyzed: 238 were women, the median age was 49.5 years [IQR 36.6-59.7]), 184 (38.9%) were intermediate risk and 51 (10.8%) with not evaluable cytogenetic and molecular data. With a median follow-up of 18.9 months, 158 (33.4%) relapsed and 130 (27.5%) died. The median (Me) OS for patients with post induction negative MRD (MRDneg) was 61.2 (CI95% not evaluable) versus 25.9 months (CI95% 2.5-49.2) with positive MRDpos, p =0.03. The RFS were 20.4 (CI95 8-32.8) and 12.4 (CI95% 6.5-18.3), p=0.06, for MRDneg and MRDpos respectively. Regarding the evaluation of MRD post C1, the Me OS was not reached for patients with MRDneg, while it was 22.1 months (CI95% 9.3-34.8) among patients with MRDpos, p=0.03. Their respective RFS were 30.3 (CI95% not evaluable) and 14.4 months (CI95% 0-30.7), p=0.03, respectively. In the subanalysis carried out in the group with intermediate or unknown cytogenetic risk post C1, the Me OS was not reached in MRDneg versus 17.5 months (CI95% 9.3-25.7) in MRDpos, p=0.007. In addition, a median RFS of 20.4 months (CI95% 0.7-40.1) vs 7.4 (CI95% 3.9-10.9), p=0.003, among those with MRDneg and MRDpos, respectively. Summary/Conclusion: The assessment of MRD by MFC showed predictive power at the different moments of follow-up evaluated. It was more evident after consolidation, with statistically significant impact in OS and RFS. The post-consolidation MRD subanalysis in the group with IR or non-evaluable cytogenetics showed promising results. This real-practice data is consistent with those published recently. Post-C1 MRD could be a useful tool in clinical practice deciding the best post-induction strategy in intermediate-risk patients. A further detailed study is required to confirm our preliminary results.

Risk Factors for Donor-Type Aplasia after Bone Marrow Transplant in Children with Acquired Bone Marrow Failure

Graft failure is a majorcause of severe aplasia after bone marrow transplant (BMT) in patients with acquired bone marrow failure (aBMF). However, we have encountered several patients with aBMF who showed persistent aplasia with full donor chimerism after BMT (donor-type aplasia). The mechanism and risk factors of donor-type aplasia are not clarified.To clarify risk factors for donor-type aplasia, we reviewed clinical data for 50 patients with aBMF who received allogeneic BMT at our hospital from August 1997 to July 2016. Out of the 50 patients, there were 32 males and 18 females. The median age at BMT was 11 years (range, 1-19 years). We retrospectively reviewed BM smears and histopathological samples at the time of BMT according to the 2008 WHO classification. Out of the 50 patients, 26 were classified as aplastic anemia (AA) and 24 as refractory cytopenia of childhood (RCC). Twenty patients received BMT from related donors including 5 human leukocyte antigen (HLA)-mismatched donors and 30 patients from unrelated donors including 15 HLA-mismatched donors. Conditioning regimens including cyclophosphamide (CY) and antithymocyte globulin (ATG) with or without low dose total body irradiation (TBI) were used for 31, fludarabine (FLU) and half dose of CY with low dose TBI were used for 15, and FLU and melphalan (MEL) with low dose TBI were used for 4 patients. Donor-type aplasia was defined as hypocellular BM with persistent cytopenia (hemoglobin <80 g/L, neutrophils <1x109/L, or platelets <50x109/L) for >6 months after achieving engraftment with full donor chimerism. To reveal risk factors of donor type aplasia, we analyzed variables as follows; age at BMT, gender, BM morphology at BMT, year performed BMT, period from diagnosis to BMT, times of red blood cell (RBC) transfusion, HLA disparity, transfused BM cell dose, conditioning regimen, graft versus host disease (GVHD), and viral infection.Out of the 50 patients, 10 patients developed late graft failure. Eight out of them developed donor-type aplasia. Out of the 8 patients, there were 5 males and 3 females. The median age at BMT was 10 years (range, 5-13 years). Bone marrow findings of all patients showed RCC at the time of BMT. Two of them received BMT from HLA-mismatched donors and 3 from unrelated donors. FLU/CY regimen was used for 6 patients, while CY/ATG regimen was used for 2 patients. All patients received low dose TBI. Three patients received BMT with <3x108/kg cell dose. Acute GVHD were observed in 2 patients and one of them developed chronic GVHD. Five patients showed cytomegalovirus antigenemia in their blood and received ganciclovir. Two patients developed Epstein-Barr virus reactivation and received rituximab. The onset of donor-type aplasia ranged from day 46 to day 710 after BMT. Five patients needed RBC or platelet transfusion, or granulocyte-colony stimulating factor (G-CSF). Donor-type aplasia continued for more than 3 years in 5 patients. Donor-type aplasia was improved spontaneously in 6 patients, ranging from 460 days to 3539 days after BMT. Only one patient underwent haploidentical stem cell transplant (SCT) from her father because of the development of severe pneumonia. She achieved engraftment after second SCT. All patients who developed donor type aplasia are alive. Cumulative incidence (CI) of donor-type aplasia was significantly higher in patients who had RCC (32%) than in patients who had AA (4%) (p = 0.013), and CI of donor-type aplasia was also significantly higher in patients who received FLU/CY regimen (44%) than in patients who received non-FLU/CY regimen (6%) (p = 0.003). Multivariate analysis confirmed that RCC (hazard ratio 8.8, 95% CI 1.1-68, p = 0.037) and FLU/CY regimen (hazard ratio 7.7, 95% CI 1.5-38, p = 0.013) were significant risk factors for donor-type aplasia.In conclusion, FLU/CY regimen is not suitable for children with RCC as a conditioning regimen of BMT. It is important to evaluate BM morphology to decide conditioning regimens for children with aBMF. The establishment of the optimal conditioning regimens for children with aBMF on the basis of the BM morphology is essential. DisclosuresNo relevant conflicts of interest to declare.

Long-Term Follow-up of Patients with Chronic Granulomatous Disease Receiving Bone Marrow Transplantation Using Immunosuppressive Conditioning Regimen

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by a mutation of one of 5 subunits of the nicotinamide adenine dinucleotide phosphate oxidase and resulting in the impairment of microbial activity of phagocytes. The disease is characterized by recurrent and severe infections of bacterial and fungal origin and a high rate of inflammatory bowel disease. Recently, the prognosis of CGD patients has been improved by the earlier diagnosis, management of infectious complications, and application of allogeneic hematopoietic stem cell transplantation (HSCT). However, allogeneic HSCT in patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality.In Hiroshima University Hospital, 38 patients with CGD underwent bone marrow transplantation (BMT) with immunosuppressive conditioning regimen since 2004. All patients with CGD suffered from recurrent and severe infections and/or intractable inflammatory bowel disease. The age of 38 patients at the time of BMT was 2 to 40 years. Six patients over 15 years old were died from GVHD and transplant-related complications.In this study we report our single-center results of long-term follow-up of 11 survivals with CGD who underwent BMT from 2004 to 2009. All 11 patients with CGD were 91-phox deficiency. The age of 11 patients at the time receiving BMT was 4 to 28 years. Seven patients were over 20 years of age, and 4 patients were 7 to 9 years, respectively. The conditioning regimen consisted of fludarabine, cyclophosphamide, 3 to 3.6 Gy of total body irradiation, melpharan, and/or antithymoglobulin. Bone marrow cells were obtained from 5 HLA-matched, 1 -mismatched related, 4 HLA-matched and 1 -mismatched unrelated donors, respectively. Acute GVHD of grade I - II developed in 4 of 11 patients. Three cases occurred in patients given transplants from unrelated donors, one from HLA-matched elder sister. All patients have exhibited 100% of complete myeloid donor chimerism and cleared all infectious and inflammatory complications. Ten of 11 patients have shown 100% of performance status without any medical supports. Four adolescent patients have been to junioror senior high school students with complete cure. Four adulthood patients fathered children 3 to 6 years after BMT. The wives of 3 patients naturally conceived, implying the successful preservation of patients' fertility. One has a child by the artificial fertilization using his frozen sperm before BMT. Only a patient who underwent BMT from HLA-matched elder sister has been suffering from chronic GVHD followed by cryptogenic organizing pneumonia. He has engaged in deskwork with 90% of Karnofsky performance status. These results suggest that BMT using immunosuppressive conditioning regimen is safe and effective for the successful transplantation followed by the improvement of quality of life for CGD patients. DisclosuresNo relevant conflicts of interest to declare.

Final Height of Patients Who Underwent Bone Marrow Transplantation for Hematological Disorders During Childhood: A Study by the Working Party for Late Effects-EBMT

AbstractFew data are available on the long-term effect of bone marrow transplantation (BMT) on growth. This study examines those factors that play a role in the final height outcome of patients who underwent BMT during childhood. Data on 181 of 230 patients with aplastic anemia, leukemias, and lymphomas who had BMT before puberty (mean age, 9.8 ± 2.6 years) and who had reached their final height were analyzed. An overall decrease in final height standard deviation score (SDS) value was found compared with the height at BMT (P < 107) and with the genetic height (P < 107). Girls did better than boys, and the younger in age the person was at time of BMT, the greater the loss in height. Previous cranial irradiation + single-dose total body irradiation (TBI) caused the greatest negative effect on final height achievement (P < 104). Fractionation of TBI reduces this effect significantly and conditioning with busulfan and cyclophosphamide seems to eliminate it. The type of transplantation, graft-versus-host disease, growth hormone, or steroid treatment did not influence final height. Irradiation, male gender and young age at BMT were found to be major factors for long-term height loss. Nevertheless, the majority of patients (140/181) have reached adult height within the normal range of the general population.

A study assessing the feasibility of randomization of pediatric and young adult patients between matched unrelated donor bone marrow transplantation and immune-suppressive therapy for newly diagnosed severe aplastic anemia: A joint pilot trial of the North American Pediatric Aplastic Anemia Consortium and the Pediatric Transplantation and Cellular Therapy Consortium.

Recent data show survival after matched unrelated donor (MUD) bone marrow transplantation (BMT) is similar to matched sibling procedures for young patients with severe aplastic anemia (SAA). Donor delays, risk of transplant-related mortality (TRM), and concern about chronic graft versus host disease raise questions about whether MUD BMT or immune suppression therapy (IST) should be preferred initial therapy for young patients lacking matched sibling donors. We performed a pilot trial to assess the feasibility of randomizing patients under age 26 with newly diagnosed SAA to receive IST versus MUD BMT. Primary aims assessed the acceptability of randomization and timing of BMT. Secondary aims measured toxicities, response, and survival. Sixty-seven patients with possible SAA were screened at nine centers. Of 57 with confirmed SAA, 23 underwent randomization and received therapy with a median follow-up of 18 months. Of 12 randomized to BMT, 10 started BMT as initial therapy at a median of 36 days after randomization. One BMT recipient experienced secondary graft failure, requiring a second procedure. Six of 11 randomized to IST responded, whereas five with refractory disease underwent successful salvage BMT. One patient achieving complete response relapsed after discontinuation of immune suppression and died of infection after salvage BMT. This feasibility study showed that a high percentage of patients underwent randomization and received up-front MUD BMT. Our study lays the groundwork for a larger randomized trial that will define best initial therapy for young patients with SAA who have an available MUD.

The Path to Cure: Using Haploidentical (haplo) Donors and High-Dose Post-Transplant Cyclophosphamide (PTCy) for Treatment-Naïve and Refractory Severe Aplastic Anemia (SAA)

SAA is a life-threatening hematopoietic stem cell disorder that is often treated with bone marrow transplantation (BMT) to reconstitute hematopoiesis and eliminate clonality. Our group has shown that PTCy after related HLA-haplo BMT can facilitate engraftment and yield low rates of relapsed and refractory (R/R) SAA. Given the risk of relapse or secondary clonal disease following immunosuppressive therapy (IST) is &amp;gt; 50% at five years, we decided to implement this treatment paradigm in untreated SAA. Here, we review the haplo outcomes in SAA, with an update in R/R SAA patients and new results in treatment-naïve (TN) SAA patients using haplo donors and PTCy. Patients were eligible with SAA. Fanconi anemia and short telomere patients were excluded. R/R patients had ≥ one course of IST. The conditioning regimen included standard published haplo backbone of antithymocyte globulin, low dose CY, and total body irradiation (TBI). All R/R patients received 200cGy. The TBI dose for the TN cohort was modified after 3 graft failures (GF) in initial 7 patients treated at 200cGy; thus, the last 8 TN patients received 400cGy. After the marrow graft was infused on day 0, cyclophosphamide 50mg/kg/day IV on days +3,+4 post-transplant was administered. All received mycophenolate mofetil beginning day +5 through 35 and tacrolimus day +5 to 365. Engraftment definitions include: absolute neutrophil count &amp;gt;1.0 x10E9/L for three consecutive days; days from last red blood cell transfusion for hemoglobin and platelet count &amp;gt;50,000 for 7 days without transfusion. Thirty-five SAA patients received haplo BMT: 20 R/R and 15 TN. The median follow-up is 31 months (90% CI: 26.0, 45.4). The median age was 25 (range, 4-69) years, with 23 patients &amp;gt; age 20 and 57% were males. The median related haplo donor age was 34 years (range, 19-57), including three non-first degree relatives. Table 1 shows data by cohort. Median time to neutrophil recovery was 17 days (range 15-88), to red cell recovery 23 days (range 6-58) and to platelet recovery 28 days (range 15-108). At the time of BMT, 25 of 35 patients had evidence of clonality (mostly PNH clones). Four of 35 patients (11.4%) (95% CI: 3.2, 26.7%) experienced GF. There was one GF out of 20 patients in the R/R group (5%) (95% CI: 0.1, 24.9%) and 3 of 15 in the TN group 20% (95% CI: 4.3,48.1%, p= 0.20). OS for all patients (Figure 1) is 94% (90% CI: 88,100%) at one and two years, 100% in the R/R group and 86% (90% CI: 72, 100%) in the TN group. The cumulative incidence of grade II-IV aGVHD at day 100 is 9% (90% CI: 1, 17%). The cumulative incidence of chronic GVHD at 2 years is 9% (90% CI: 1,17%). All R/R patients are alive and well, and fully engrafted with 100% donor chimerism; patient with primary GF is 100% chimeric after 2nd transplant from different haplo donor. One patient has extensive cGVHD. Of the first 7 TN patients who received 200cGy TBI, 4 are fully engrafted and well; of the 3 with GF, 2 died from infection and one is now 100% chimeric using second haplo donor. After increasing the TBI dose to 400cGy, the subsequent 8 patients are alive with full donor chimerism. Nonmyeloablative haplo BMT with PTCy represents a potential cure in SAA, with all 20 R/R currently alive, disease-free (including eradication of clonal diseases), and no evidence of active GVHD. Extending this approach to TN patients was associated with higher GF rates. Perhaps this is not surprising given that the potential beneficial effect of front-line IST on engraftment is absent. The increase in TBI dose to 400cGy in TN patients appears to achieve more durable engraftment without early greater toxicity (infection, delayed engraftment, GVHD); more patients and longer follow up are required to understand rates of fertility and other survivorship issues. Alternative donor BMT (including haplo donors) should be offered to all SAA patients with R/R SAA who are fit enough to tolerate non-myeloablative conditioning. A potential advantage of haplo donors over unrelated donors is that the transplant center has full control and oversight of allograft quality, which can be especially important with the use of the preferred allograft source for SAA, bone marrow. Non-myeloablative haplo BMT in TN SAA could lead to a paradigm-shift in the field, such that essentially all children and fit adults can proceed quickly to a relatively safe, curative BMT. Disclosures DeZern: Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Cooke:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

P4.60: Bortezomib for the treatment of chronic Graft Versus Host Disease in Intestinal Transplant recipient

Introduction: GVHD is a disparate immunological response between host and recipient tissues. When it occurs after Intestinal Transplantation (ITx), it can be deadly. Bortezomib is a proteasome inhibitor that affects dendritic and T-cell processes essential for the development of GVHD but also decreases B cells. The use of Bortezomib at time of bone marrow transplant (BMT) in mice protected against acute GVHD. Bortezomib has been used in ITx for refractory acute rejection with donor specific antibodies. To our knowledge, we present the 1st use of Bortezomib for chronic GVHD in ITx. Case: 25 yo female with Pseudo-obstruction as a result of Ehlers Danlos received a modified MVT. Induction immunosuppression (ISP) was Antithymoglobulin/Rituximab/Methylpred/Basiliximab and maintenance FK/Everolimus. Blood chimerism was followed serially. She developed a skin rash and persistent fevers on week (wk) 4 post transplant. Chimerism rose from 33% wk 4, 61% wk 5 and 80% wk 6. Endoscopy on wk 5 was negative for rectal chimerism and GHVD. Given ongoing symptoms, Alemtuzumab 30 mg IV x 2 was given for acute GVHD. Chimerism decreased to 1% wk 8 but again rose to 96% wk 11, fluctuating over the next month, 56–96%. A 3rd dose of Alemtuzumab was given wk 15. Chimerism decreased to 2% by 5 months (mn) post transplant. 6 mn post transplant, she developed neutropenia. Given possible bone marrow GVHD, a biopsy was done and negative. She again developed rash with alopecia but managed conservatively with prednisone. Course complicated with BK viremia (5.6 million copies) treated empirically with IVIG and decrease in ISP. 9 mn post transplant, she lost 14 pounds due to high ostomy output. Work up ruled out rejection, allergy and infection except BKV pcr + in bowel biopsy. She started Cidofovir, symptoms improved and blood BKV pcr decreased. 12 mn post transplant, liver enzymes increased and biopsy ruled out GVHD. However, she again presented with skin rash despite chimerism <5%. 14 mn post transplant, Bortezomib was given weekly x 4 for chronic GVHD. Thereafter, rash resolved in 4 weeks with no recurrence now 2 years post transplant. Discussion: GVHD continues to be a threat to ITx recipients with high mortality. Diagnosis is notoriously difficult as biopsies tend to be negative early in the course. A strong index of suspicion is necessary, before life threatening organ involvement develop, as early treatment is key to survival. Bortezomib may be an option for chronic GVHD in ITx.

A Pilot Study of Human Milk to Reduce Intestinal Inflammation After Bone Marrow Transplant.

Human milk administration in the early peritransplant period would lower intestinal inflammation after bone marrow transplant (BMT). Children 0-5 years undergoing BMT received either a ready-to-feed human milk preparation designed for these children (Prolacta Bioscience, Duarte, CA) or standard formula. Babies breastfeeding at the time of BMT were also enrolled on the human milk arm. Human milk was administered from day -3 until day +14 after BMT. Metagenomic shotgun sequencing and metabolomics of stool, plasma cytokines, and regenerating islet-derived 3α (REG3α) levels were measured at enrollment and day +14. Human leukocyte antigen-DR isotype (HLA-DR), CD38, and CD69 expression on T cells were evaluated at day +21. Forty-six children were enrolled, 32 received human milk (donor milk n = 23, breastfeeding babies n = 9), and 14 were controls who received standard feeds supervised by a BMT dietician. Twenty-four patients received at least 60% of goal human milk and were evaluable. Plasma interleukin (IL)-8 (p = 0.04), IL-10 (p = 0.02), and REG3α (p = 0.03) were decreased in the human milk cohort. Peripheral blood CD69+ CD8+ T cells were higher in controls (p = 0.01). Species abundance of Adenovirus (p = 0.00034), Escherichia coli (p = 0.0017), Cryptosporidium parvum (p = 0.0006), Dialister invisus (p = 0.01), and Pseudomonas aeruginosa (p = 0.05) from stool was higher in controls. Stool alanine, tyrosine, methionine, and the ratio of fecal alanine to choline and phosphocholine were higher in controls (p < 0.05). No difference was observed in stool propionate and butyrate levels as measures of short-chain fatty acids between the two cohorts. Administration of human milk resulted in decreased markers of intestinal inflammation and could be a valuable adjunct for patients after BMT.

Patients with TTC7A Deficiency Causing Multiple Intestinal Atresia with Combined Immunodeficiency (MIA-CID) Can Survive Well Beyond Infancy

Introduction: TTC7A gene mutations have recently been identified as a cause of Multiple Intestinal Atresia with Combined Immunodeficiency (MIA-CID), an extremely rare condition with few patients surviving beyond infancy. This case series reports the clinical characteristics of six surviving children. Methods: Records of all children with MIA-CID and TTC7A mutations confirmed on genetic testing at two tertiary intestinal rehabilitation centres were reviewed retrospectively to identify common phenotypic findings and current management strategies. Results: Six patients (three female) were identified. All patients had multiple intestinal atresias requiring early surgical intervention. All patients required additional operations for acquired strictures. Mean length of remaining small bowel in continuity was 8 cm (range 4 to 20 cm). Only one patient was able to tolerate any substantial enteral feed; all patients required lifelong parenteral nutrition, except one, who achieved full enteral autonomy after a multivisceral transplantation at five years of age. Four patients received bone marrow transplants with successful engraftment as treatment of their immunodeficiency, which was characterized primarily by severe T-cell lymphopenia. All patients developed intestinal failure associated liver disease. One child had a multivisceral transplantation, a second is listed and a third is undergoing evaluation for multivisceral transplantation. The other three have stable liver disease on non-soybean-based lipid formulations. There is variable occurrence of pulmonary, dermatological and cardiac disease in our cohort. Conclusions: Patients with TTC7A deficiency leading to MIA-CID can survive beyond infancy with intensive surgical and multidisciplinary medical management. Patients develop intestinal strictures requiring proximal stomas and are unable to establish enteral autonomy leading to parenteral nutrition dependence. Intestinal failure associated liver disease is seen in all patients. The optimal timing of bone marrow transplantation requires consideration, especially for patients considering multivisceral transplantation and for those who do not have a fully matched donor.

Combining Flow Cytometry and Molecular Assessment Improves the Prognostic Value of Pre-Transplant Minimal Residual Disease in Acute Myeloid Leukemia

BACKGROUND AND AIMSAllogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for high-risk acute myeloid leukemia (AML) patients. Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. Multicolor flow cytometry (MFC) is the most common tool to evaluate MRD, whereas WT1 gene expression is the most widely applicable and standardized molecular MRD marker in AML. The aim of this study was to analyze the role of pre-BMT combined MFC-molecular MRD assessment as predictor for the post-transplant relapse risk.MATERIALS AND METHODSWe retrospectively analyzed the outcome of 224 consecutive AML patients receiving allo-BMT in 1st or 2nd CR. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation. Median age at transplant was 44 years. Disease phase was CR1 in 161 (72%) and CR2 in 63 patients (28%). BMT conditioning wasmyeloablative in 163 (73%), whereas 61 patients (27%) received reduced intensity conditioning. Stem cell source was HLA-identical sibling in 79 (35%),haploidentical in 59 (27%) and alternative donor in 86 (38%).Median follow-up was 64 months (95% CI 50.3 - 77.3 months). Cumulative Incidence of Relapse (CI of relapse) at various time-points was calculated in competing risk analysis by takingin account non relapse mortality as competing event. Overall Survival (OS) was calculated from the time of BMT until death by any cause or last follow-up.A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression.RESULTSRelapse occurred in 62 patients (27.7%). Three-year estimate of CI of relapse was 25.9% (median not reached). The CI of relapse was significantly affected by occurrence of acute GVHD (lower for grade ≥2, p <0.05) and MRD status before BMT, measured with any method (p <0.001 for WT1-based MRD, p<0.03 for MFC based MRD, p<0.0001 for combined MRD). In multivariate analysis the combined MRD evaluation and the onset ofaGVHDwere the only independent predictor of CI of relapse (p <0.001 and 0.04, respectively, Table I).Specifically, patients with double positive molecular and MFC MRD had a very poor outcome, with a 3-year CI of relapse of 51.9% (Figure 1). On the contrary, patients with negative combined MRD had a very low CI of relapse, regardless of the conditioning regimen received, whereas a trend toward benefit frommyeloablativeconditioning was observed in patients with MFC positive/WT1 negative MRD and double positive patients.Patients with negative MFC-MRD undergoing BMT in 1st or 2nd CR had a very similar outcome (3-year CI of relapse 8.3 and 5.6%, respectively). Similarly, MFC positive/WT1 negative MRD patients had a 3-year CI of relapse of 17.3 and 18.% for CR1 and CR2 patients, respectively. Patients who were MRD positive by both methods undergoing BMT in CR1 or CR2 had a similar, very high CI of relapse (Figure 2).MRD evaluation was also a strong predictor of long term survival: 3- years OS was 73.5% for MFC negative and 36.7% for double WT1 and MFC MRD positive patients, respectively, (p <0.001). Multivariate OS analysis showed that conditioning intensity and combined MRD evaluation significantly influenced OS duration (p <0.005 and <0.001, respectively)CONCLUSIONSPre transplant MRD evaluation by combined molecular and MFC assessment on bone marrow samples is a reliable predictor of relapse risk. Patients with both negative pre-BMT MRD markers have a significantly lower CI of relapse, while patients with both positive MRD markers display an higher risk of relapse. Given the high prognostic value, combined molecular/MFC pre-transplant MRD status may be taken in account when considering the most appropriate stem cell source or conditioning for each patient. Furthermore, identifying patient in morphological CR at BMT but with a very high risk of relapse, such as double positive patients, could open the way to apply pre-emptive therapeutic strategies to prevent AML relapse, from donor lymphocyte infusion to other innovative approaches. [Display omitted] [Display omitted] [Display omitted] DisclosuresGobbi:Janssen: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Celgene: Consultancy; Roche: Honoraria; Mundipharma: Consultancy, Research Funding; Takeda: Consultancy; Gilead: Honoraria.

Protection of Factor VIII-Specific IgG Antibody Response and IgE Formation By Pretreatment with Liposomal α-Galactosylceramide in the Enzyme Replacement Therapy Mouse Model

▪Enzyme replacement therapy (ERT) is indispensable for patients of hemophilia or lysosome storage disease (LSD) lifetime. However, the efficacy of ERT is diminished or lost in some patients who acquired the immune responses by the repeated enzyme infusions. If reduced the immune responses such as neutralizing IgG antibody formation and/or anaphylaxis, one enzyme for ERT would be continuously administered without loss of efficacy and/or anaphylaxis risk. We here show that the pre-treatment with α-galactosylceramide (αGC), a representative ligand for invariant natural killer T (iNKT) cells prior to the ERT could remarkably suppress not only the enzyme-specific IgG antibody response but also total IgE formation in a mice model.In our previous studies, liposomal, but not aqueous, αGC formulations were delivered to the splenic marginal zone (MZ) B cells which could enhance IL-10 production after the interaction with iNKT cells, and then the expansion of tolerogenic dendritic cells (DCs) and Foxp3-positive regulatory T cells (Tregs) (Ishii et al. Front Biosci 2008). The pretreatment with a liposomal αGC (lipo-αGC) formulation resulted in the remarkable suppression of both IgG and IgE antibody responses in mice immunized with alum-absorbed ovalbumin. In addition, administration of RGI-2001, a clinical study grade of lipo-αGC formulation, at the timing of bone marrow transplantation could induce the expansion of tolerogenic DCs and/or allo-specific Tregs in mouse models of either acute graft versus host disease (GvHD) or the mixed chimera formation (Duramad et al. Biol Blood Marrow Transplant 2011, Hirai et al. Am J Transplant 2014 and Hirai et al. Am J Transplant2016). Taken together, it was suggested that administration of lipo-αGC formulations prior to the ERT could work for the protection of the enzyme-specific antibody responses.To test the suppressive capability of a lipo-αGC for ERT application, a mouse system to monitor a factor VIII-specific IgG antibody titer and IgE level in bloods was established. In BALB/c mice given the repeated intravenous injections of recombinant factor VIII, the factor VIII-specific IgG titer and total IgE concentration were substantially elevated (ERT-mice). The mice pretreated with the intravenous single injection of the lipo-αGC showed that not only the factor VIII-specific IgG but also total IgE was significantly suppressed compared to the untreated control mice. The antibody-suppressive efficacy by multiple injection of the lipo-αGC was comparable with that by single injection in the ERT-mice. However, cytokine profile of splenic iNKT cells was dramatically changed in BALB/c mice pretreated with either single or multiple lipo-αGC injections. The iNKT cells derived from mice pretreated with the single the lipo-αGC injection expressed high level of IFN-γ, IL-4 and IL-13 mRNAs but not IL-10 mRNA. Conversely, the iNKT cells pretreated with multiple the lipo-αGC injections expressed higher level of IL-10 mRNA more than IFN-γ mRNA, but remarkably diminished both IL-4 and IL-13 mRNAs. As known that αGC-activated iNKT cells could rapidly produce cytokines within a few hours, we then examined the last injection timing of multiple injections into the ERT-mice. The last injection at the timing of the ERT start did not show stronger efficacy than that at three day before the ERT start. Results collectively suggested that the suppression of IgG/IgE formations in the ERT-mice might not be caused by high IL-10 expression of iNKT cells by pretreatment with multiple the lipo-αGC injections, but other phenomena generated in three days after the interaction of activated iNKT cells with αGC-presenting MZ B cells.We conclude that the lipo-αGC pretreatment is capable of protecting harmful formation of both the enzyme-specific neutralizing IgG and anaphylaxis-inducing IgE antibodies during ERT. DisclosuresNo relevant conflicts of interest to declare.

Chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by recurrent, life-threatening bacterial and fungal infections of the skin, the airways, the lymph nodes, the liver, the brain and the bones. Frequently found pathogens are Staphylococcus aureus, Aspergillus species, Klebsiella species, Burkholderia cepacia, Serratia marcescens and Salmonella species. CGD is a rare (∼1:250 000 individuals) disease caused by mutations in any one of the five components of the NADPH oxidase in phagocytic leucocytes. This enzyme generates superoxide and is essential for intracellular killing of pathogens by phagocytes. CGD patients suffer not only from life-threatening infections, but also from excessive inflammatory reactions. Neither the cause of these inflammatory reactions nor the way to treat them is clear. Patient selection for and timing of bone marrow transplantation along with gene therapy.

IL-2 / α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment

Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM rejection, as their effect is not sufficiently restricted to Tregs but rather extends to several other lymphocyte populations.

A Multicenter Phase II Trial of Unrelated Donor Reduced Intensity Bone Marrow Transplantation for Children with Severe Sickle Cell Disease (SCURT): Results of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN 0601) Study

▪Background:Children with severe sickle cell disease (SCD) experience organ damage, poor quality of life, and a risk of premature mortality. Allogeneic bone marrow transplantation (BMT) halts disease progression when stable donor erythropoiesis is established. Myeloablative conditioning (MAC) followed by HLA matched sibling donor BMT is effective, but most patients with SCD lack a matched sibling donor and long term side effects from MAC (e.g., infertility) can be barriers to patient acceptance of BMT. The SCURT trial was designed as a prospective, multicenter phase II trial to evaluate outcomes after HLA matched unrelated donor (MUD) BMT with reduced intensity conditioning (RIC). We hypothesized that an intensely immunosuppressive RIC regimen would be sufficient for donor engraftment and could limit toxicity. The primary endpoint was one year event free survival (EFS) (alive and disease free) ≥75%. All patients were evaluable for the primary endpoint for the purpose of this report.Methods:Children between 3 and 19.75 years of age who had ≥1of the following complications - stroke, high transcranial doppler velocity, recurrent acute chest syndrome, or significant vaso-occlusive pain crises were eligible. An independent review committee confirmed eligibility. The preparative regimen consisted of alemtuzumab (48 mg) (day-22 to -19), fludarabine (150 mg/m2) (day-8 to -4), and melphalan (140mg/m2) (day-3). Donors and recipients were matched at the HLA allele-level at HLA-A, -B, -C and -DRB1 loci. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus or cyclosporine, methotrexate (7.5 mg/m2)on days +1, +3 and +6, and methylprednisolone (1 mg/kg/day) from day +7, tapered after day +28.Results:Thirty patients were enrolled between 2008 and 2014. One patient was excluded from analysis for a major eligibility violation at the DSMB's recommendation. Twenty-nine patients (16 M, 13F), 5.9-19.3 years old (median 14.0 years) from 19 centers were evaluable with median follow up of 25.2 months (range, 12-61.5 months). All patients recovered neutrophils (median 12 days) and platelets >50x109/L (median 24 days). Graft rejection (GR) in 3 patients (10%) occurred before day +100, and was followed by autologous hematopoietic recovery. No late GR has been observed. The primary endpoint was met with1-year EFS of 76% (Figure 1A). The 1-year probability of overall survival (OS) was 86% (Figure 1B). The cumulative incidence (CINC) of grade II-IV and III-IV acute GVHD at day 180 were 31% and 17% respectively (Figure 2A). The CINC of chronic GVHD at 1-year was 62%. Chronic GVHD was extensive in 38% (Figure 2B). GVHD when present was noted by 12 months post-BMT. There were 7 deaths, all in patients >14 years at time of BMT. One death occurred after a second transplant for graft rejection. The other 6 deaths occurred between 143 and 960 days post BMT, from GVHD. Non-fatal toxicities included posterior reversible encephalopathy syndrome (PRES) associated with hypertension and seizures in 35%. All cases were observed in the first 6 months post-BMT; all patients recovered with no obvious sequelae. No patient developed veno-occlusive disease, idiopathic pneumonia syndrome or cerebral hemorrhage. Infections were common, and occurred in 76% of patients. CMV and EBV reactivation each occurred in 23% of recipients. Surviving patients report performance scores of 100% (n=14), 90% (n=5), and 80% (n=3) at 2 years or at the time of last assessment. At last follow up, 13 patients had successfully discontinued immunosuppressive therapy.Conclusion:In summary, the RIC regimen employed in this trial of MUD BMT in children with severe SCD resulted in low rates of regimen-related organ toxicity (other than a high rate of PRES) and 1-year EFS of 76%, associated with regression of SCD manifestations. . The risk of graft rejection/disease recurrence was similar to that observed after conventional HLA-identical sibling donor BMT. Chronic GVHD occurred in 62% of patients and was the leading cause of toxicity and mortality. Future trials should focus on effective GVHD prophylaxis after URD BMT. Secondary outcome analyses including health-related quality of life and neurocognitive performance are ongoing. [Display omitted] [Display omitted] DisclosuresWalters:ViaCord and AllCells, Inc: Other: Medical director. Levine:Novartis: Consultancy.

Using Haploidentical (haplo) Donors and High-Dose Post-Transplant Cyclophosphamide (PTCy) for Refractory Severe Aplastic Anemia (SAA)

SAA is a life-threatening hematopoietic stem cell disorder that is treated with either bone marrow transplantation (BMT) or immunosuppressive therapy (IST). BMT is the treatment of choice for young patients with a matched sibling donor. Those with acquired SAA lacking a matched sibling donor and older adults (> 40yrs) generally receive IST because of the high toxicity of BMT in these patients. Roughly two-thirds will respond but up to 40% of patients eventually relapse or acquire a secondary clonal disease such as paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplastic syndromes. Thus, a major challenge in treating SAA is the management of patients who are have relapsed after or are refractory to IST or who have acquired a secondary clonal disorder. BMT is the only curative therapy for patients with inherited SAA but problems with graft failure, graft-versus-host disease (GVHD), and lack of matched unrelated donors especially for ethnic minorities have limited the success of alternative donor BMT, especially for adult patients. PTCy after HLA-haplo BMT has been shown to facilitate engraftment and yield rates of GVHD comparable to those of matched sibling BMT in hematologic malignancies. We hypothesized that PTCy would improve outcomes for patients with refractory SAA by reducing the risk of GVHD and expanding the donor pool to include haplo related and mismatched unrelated donors (URD).Patients received an allogeneic BMT (alloBMT) for relapsed or refractory SAA. Relapsed/ refractory disease was defined as having failed previous course of IST. In the majority of the cohort, haplo donors were used due to lack of availability of suitable and timely URDs. Nine patients underwent haploBMT. A mismatched URD was used in one case of no suitably available haplo donor given the inherited nature of her disorder. The nonmyeloablative preparatory regimen included antithymocyte globulin (0.5 mg/kg day-9 and 2 mg/kg on days -8,-7), fludarabine (30mg/m2/day on days - to -2), low dose CY (14.5 mg/kg on days -4, -5 ) and total body irradiation (200cGy day -1). After the unmanipulated marrow graft was infused on day 0, cyclophosphamide 50mg/kg/day IV on days +3,+4 post-transplant was administered. All patients received tacrolimus and mycophenolate mofetil (MMF) beginning day+5 through 35 (MMF) and day +5 through 1 year (tacrolimus) for additional graft versus host disease (GVHD) prophylaxis. Neutrophil engraftment was defined as an ANC >1.0 x109/L measured for three consecutive measurements on different days. Red cell engraftment was defined as days from last red blood cell transfusion. Platelet engraftment was defined as a platelet count >50,000 for 7 days without transfusion.Ten patients were treated according to this IRB approved protocol. Eight patients had documented acquired disease and received grafts from 5/10 related donors. Six had failed ATG-containing regimens previously and two relapsed after high dose cyclophosphamide as IST. Two patients had inherited syndromes: one with Diamond Blackfan who received a graft from 5/10 related donor and one with telomeres less than the first percentile in length with a presumed familial syndrome who received a 9/10 unrelated graft. Median follow-up time is 17.5 months (range 5-49). The median age was 34 years (range 17-54) with 6 patients greater than age 30 years, and 50% were males. Median time to neutrophil engraftment was 18 days (range 16-24). Median time to red cell engraftment was 25 days (range 16-48). Median time to platelet engraftment was 27.5 days (range 22-108). At the time of BMT, six patients had PNH clones, and all were eliminated. All patients are alive and well, fully engrafted with 100% donor chimerism in blood and marrow. Two patients had grade 1-2 skin acute GVHD. These two also had mild chronic GVHD of the skin/mouth requiring systemic steroids. One patient was able to come off all immunosuppression by 15 months and the other by 17 months.Nonmyeloablative alloBMT with PTCy appears promising in patients with refractory acquired and inherited SAA with acceptable rates of engraftment, eradication of pre-existing clonal diseases, and expansion of the donor pool. Given these promising results, this approach is being utilized in the next BMTCTN national trial for acquired refractory SAA. We believe this approach could also be considered as upfront therapy for SAA, and such a trial is in development at Hopkins DisclosuresBrodsky:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Ocular Chronic Graft Versus Host Disease after Bone Marrow Transplantation

PurposeAllogeneic bone marrow transplantation (BMT) is a curative therapy for a number of hematological diseases. Ocular chronic graft versus host disease (cGVHD) is a major contributor to long‐term morbidity after BMT. The purpose of this study was to report the frequency of ocular cGVHD after BMT and onset in relation to systemic cGVHD.MethodsRetrospective examination of medical records of patients who underwent consecutive allogeneic BMT from 1980‐2011 at Copenhagen University Hospital (Rigshospitalet). This study included adults (&gt;16 years) with no dry eye disease prior to BMT. The patients were seen by an ophthalmologist before BMT and annually after BMT. The ophthalmological examination included tear break‐up time, Schirmers test, corneal fluorescein stain, slit lamp examination and ophthalmoscopy. The criteria proposed by the “International Chronic Ocular GVHD Consensus Group” was used to diagnose ocular cGVHD.ResultsOut of the 939 patients included, 222 patients (23.6%) developed ocular cGVHD. We found no significant difference between gender and the development of ocular GVHD (p = 0.67). Median age at time of BMT was 41 years (rage 16–73) and 46 years (17–68) in the group who developed ocular cGVHD. Median time of onset of ocular cGVHD was 20 months (0.4–196) after BMT. Diagnosis of ocular cGVHD preceded systemic cGVHD in 33 cases (15%). Twenty‐seven patients (12%) had ocular cGVHD without systemic cGVHD. Ocular cGVHD was significantly higher in patients with systemic cGVHD involving the skin (p &lt; 0.001).ConclusionsOcular cGVHD is common after BMT. It can occur at any age, but is more common in elder patients. Ocular cGVHD can occur in patients without systemic cGVHD. We recommend ophthalmological examinations in all patients before and after allogeneic BMT due to the high frequency of ocular cGVHD.

Acute graft-versus-host disease impairs anti-viral immunity and leads to fulminant CMV disease (TRAN1P.937)

Abstract Viral infection is a common, life-threatening complication of allogeneic bone marrow transplantation (BMT), particularly in the presence of graft-versus-host disease (GVHD). The inability to mount protective anti-viral immunity is well recognized, yet the defects remain unclear. Using cytomegalovirus (CMV) as the prototype pathogen, we delineated the cause of the disease that arises following infection, as well as the mechanisms that lead to the inability to mount effective anti-viral immunity in this setting. While infection was self-limiting after syngeneic BMT, CMV infected mice with GVHD developed a fulminant necrotizing hepatitis directly associated with extensive CMV cytopathy. GVHD induced profound defects in dendritic cells (numerical and resistance to infection) that profoundly impaired viral antigen presentation, preventing the priming of CMV-specific CD8+ T cells. Changes indicative of T cell exhaustion were also observed. The transfer of virus-specific CD8+ T cells pre-infection circumvented the priming defect and provided protective immunity. Notably, efficacious anti-viral immunity could still be generated when T cells were transferred at the time of BMT, a month prior to infection, despite concurrent GVHD. These data demonstrate the importance of avoiding GVHD when reconstructing anti-viral immunity after BMT, and highlight the mechanisms by which the adoptive transfer of virus-specific T cells overcome the endogenous defects in priming invoked by GVHD.

Graft-Versus-Host Disease (GVHD) and Survival Outcomes after HLA-Haploidentical (Haplo) Bone Marrow Transplant (BMT) Compare Favorably with Matched Related Donor (MRD), and Matched Unrelated Donor (MUD) BMT Utilizing High-Dose Posttransplantation Cyclophosphamide (PTCy)

Background: High-dose PTCy has been shown to effectively reduce acute (a) and chronic (c) GVHD after MRD and MUD BMT and to enable the safe implementation of haplo BMT. However, GVHD-related risk factors and survival outcomes, the impact of donor type, and novel composite endpoints, such as cGVHD-free relapse-free survival (cGFRFS), have not been fully characterized in BMT with PTCy.Methods: We retrospectively analyzed 582 consecutive adult pts with advanced or poor-risk hematologic malignancies who received allogeneic BMT with PTCy at Johns Hopkins from 2002-2012. All pts received a T-cell replete bone marrow graft and PTCy (50 mg/kg/d IV) on D+3 and +4. Platforms consisted of: 1) myeloablative conditioning (MAC) with Busulfan/Fludarabine or Busulfan/Cy followed by MRD (n = 193; 33%) or MUD (n=119; 20%) allografting and PTCy as sole GVHD prophylaxis; or 2) nonmyeloablative (NMA) conditioning with Fludarabine/Cy/TBI followed by related haplo (n = 270; 46%) allografting and PTCy, mycophenolate mofetil D5-35, and tacrolimus D5-180.Results: The median follow up was 4 (range 0.3-11.4) years (y). The median pt age at time of BMT was 49 (range 18-66) in the MAC cohort and 54 (range 18-73) in the NMA cohort. On competing risk analysis, the D200 probability of grade II-IV aGVHD was 27% after haplo, 37% after MRD, and 50% after MUD BMT, and grade III-IV aGVHD was 4%, 11%, and 14% respectively. The 3-y probability of cGVHD was 12% after haplo, 8% after MRD, and 19% after MUD BMT. On multivariate analysis adjusted for age and original disease risk index (DRI), MRD and MUD BMT were associated with a statistically significantly higher risk of grade II-IV and grade III-IV aGVHD compared to haplo BMT (each p ≤ 0.001). Compared to haplo BMT, MUD, but not MRD, BMT was also associated with a significantly higher risk of cGVHD (p = 0.009). On multivariate analysis, female into male allografting was independently associated with a higher risk of cGVHD (p = 0.007), whereas age and CMV serostatus of the pt and donor were not statistically significantly associated with risks of acute or chronic GVHD. Pts with grade II-IV aGVHD or cGVHD had a lower probability of relapse, but a higher probability of nonrelapse mortality (NRM) at 3 y compared to pts without GVHD. Among pts with GVHD, an HCT-CI score ≥ 5 was associated with significantly inferior overall survival compared to lower HCT-CI scores. Although MAC matched BMT was associated with less relapse risk than NMA haplo BMT, the 3-y probabilities of NRM, DFS, and overall survival were comparable between the transplant platforms (Table). On multivariate analysis adjusted for pt age and DRI, there were no statistically significant differences in DFS, OS, or cGFRFS in MAC related or unrelated BMT compared to NMA haplo BMT.Conclusions: High-dose PTCy safely modulates acute and chronic GVHD across multiple BMT platforms. The apparently lower risk of aGVHD after NMA haplo BMT with PTCy likely reflects the reduced conditioning intensity and longer duration of immunosuppression utilized. Despite the limitations inherent to a retrospective analysis, these data suggest that key outcomes after BMT with PTCy are comparable across donor types and conditioning intensities. Notably, in all these settings, 3-y estimates of cGFRFS appeared to approach those of DFS. Thus, PTCy may minimize the late morbidity and mortality of cGVHD and allow earlier implementation of novel posttransplantation strategies for relapse prevention. TableOutcomes of BMT with PTCy3-y probabilitiesNMA HaploMAC MRD*MAC MUD*NRM141717DFS384144OS495856cGFRFS313533* p = NS for all comparisons to haplo BMT on multivariate analysis DisclosuresOff Label Use: High-dose posttransplantation cyclophosphamide.

Clinical Impact of Radiation-Resistant Mesenchymal Stem Cells in Bone Marrow Deduced from Preclinical Studies

Mesenchymal stem cells in the bone marrow have attracted great interest over the past decades, not only as a basic scientific subject but also as a novel and advanced clinical tool. More than 100 mesenchymal stem cell-related clinical trials are currently registered in the world. Hematopoietic stem cells in bone marrow are extremely radiationsensitive, whereas mesenchymal stem cells show considerable radiation-resistance. Intrinsic cellular mechanisms, including highly efficient reactive oxygen species-scavenging ability and active DNA damage response pathways, have been reported to explain the radiation-resistance of mesenchymal stem cells in the bone marrow. The precise interactions between residual host mesenchymal stem cells and donor mesenchymal stem cells at the time of bone marrow transplantation following whole-body irradiation, however, remain unknown. This short review summarizes our current understanding of the clinical impact of the radiation-resistance of endogenous mesenchymal stem cells in the bone marrow.