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Highlights • The impact of donor age and sex but not CMV matching is comparable with that of single HLA disparity on survival after HSCT. • Similar survival after matched unrelated and related donor HSCT for AML or MDS reflects lower NRM but higher relapse risks in the latter. Abstract Increasing donor–recipient HLA disparity is associated with negative outcomes of allogeneic hematopoietic stem cell transplantation (HSCT), but its comparative relevance amid non-HLA donor characteristics is not well established. We addressed this question in 3215 HSCTs performed between 2005 and 2013 in Germany for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Donors were HLA-matched related (MRD; n = 872) or unrelated (10/10 MUD, n = 1553) or HLA-mismatched unrelated (<10/10 MMUD, n = 790). Overall survival (OS) was similar after MRD compared with 10/10 MUD HSCT, reflecting opposing hazards of relapse (hazard ratio [HR], 1.32; P <.002) and nonrelapse mortality (HR,.63; P <.001). After UD HSCT, increasing HLA disparity was associated with inferior OS (HR, 1.21 [ P <.02] and HR, 1.57 [ P <.001] for 9/10 and ≤8/10 MMUD, respectively, compared with 10/10 MUD). Among non-HLA donor characteristics, age, sex mismatching (male recipient–female donor), and cytomegalovirus (CMV) mismatching (positive recipient–negative donor) impacted OS. Multivariate subgroup analysis showed that OS was similar after HSCT from the youngest 9/10 MMUD (<30 years) compared with the oldest 10/10 MUD (>40 years) (HR, 1.18; P =.25) and also in male patients transplanted from female 10/10 MUD compared with male 9/10 MMUD (HR,.89; P =.46). In contrast, OS of CMV-positive patients tended to be better with CMV-negative 10/10 MUDs compared with CMV-positive 9/10 MMUDs (HR, 1.31; P =.04). Because of low patient numbers in subgroups, definite conclusions and establishment of a hierarchy among HLA matching and non-HLA donor characteristics could not be made. Our data suggest that the impact of donor age and sex mismatch but not CMV mismatch on outcome of allogeneic HSCT may be comparable with that of single HLA disparity. [ABSTRACT FROM AUTHOR]

Since 2000, various phase III randomized controlled trials (RCTs) have investigated the efficacy of rabbit antithymocyte globulin (ATG) in patients following allogeneic stem cell transplantation (allo-SCT). Comparisons of different ATG formulations are lacking, however. Our aim was to synthesize all published efficacy evidence to enable a comparison of all available formulations of rabbit ATG in the allo-SCT setting. We performed a systematic literature review to identify all available phase III RCT evidence. We searched the Cochrane Library, MEDLINE, MEDLINE In-Process, and the website www.ClinicalTrials.gov . In addition, a trial presented at the Annual Meeting of the American Society of Hematology 2016 was added to include the most recent evidence. We identified a total of 6 RCTs, including 2 formulations: anti–T lymphocyte globulin (ATLG; Grafalon, Neovii Biotech, Lexington, MA) and polyclonal globulin immunized with human thymocytes (Thymoglobulin [Thymo]; Genzyme-Sanofi, Cambridge, MA). The evidence was synthesized using a conventional network meta-analysis (NMA). The best treatment for preventing graft-versus-host disease (GVHD) was ATLG, which had a more favorable hazard ratio (HR) compared with standard treatment (chronic GVHD: HR, .42; 95% confidence interval [CI], .31 to .56; acute GVHD grade II-IV: HR, .54; 95% CI, .39 to .73; acute GVHD grade III-IV: HR, .50; 95% CI, .29 to .86), whereas both ATLG and Thymo were at least similarly effective in terms of transplantation-related mortality (TRM) (ATLG: HR, .90; 95% CI, .61 to 1.32; Thymo: HR, .90; 95% CI, .56 to 1.44). Thymo tended to be the better treatment option regarding overall survival (OS) (HR, .86; 95% CI, .59 to 1.26). Our NMA provides the first report of the relative efficacy of all available rabbit ATG formulations in patients undergoing allo-SCT. Until additional data from randomized head-to-head comparisons are available, based on the present analysis, ATLG seems to be the best option to prevent chronic and acute GVHD. Both formulations show similar efficacy in terms of TRM, whereas Thymo appears to be the better treatment option in terms of OS. [ABSTRACT FROM AUTHOR]

Abstract: To assess current clinical practice in diagnosis and treatment of acute graft-versus-host disease (aGVHD), we performed a survey among German, Austrian, and Swiss allogeneic hematopoietic stem cell transplantation (allo-HSCT) centers. Thirty-four of 72 contacted centers (47%) completed both the diagnostic and therapeutic sections of the survey, representing 65% of allo-HSCT activity within the participating countries in 2011. Three pediatric centers answered as requested only the diagnostic part of the survey. In the presence of diarrhea and decreased oral intake after engraftment, only 4 centers (12%) do not perform any endoscopy before the start of immunosuppressive treatment. In case of a skin rash with the differential diagnosis of drug reaction, only 12 centers (35%) perform a skin biopsy up front, whereas 19 do so after failure of systemic steroids. In the presence of rapidly increasing cholestasis occurring without any other signs of aGVHD, 11 centers (32%) perform a liver biopsy up front and 14 only after failure of steroid treatment, whereas 9 centers do not perform a liver biopsy at all. Twenty centers (59%) use a percutaneous approach, 12 a transvenous approach, and 1 mini-laparoscopy for liver biopsies. First-line treatment of cutaneous aGVHD stage 1 consists of topical treatment alone in 17 of 31 responding centers (61%), whereas isolated cutaneous aGVHD stage III is treated with systemic steroids (prednisolone below 0.5 mg/kg/day n = 2, 0.5 to 1.0 mg/kg/day n = 10, above 1.0 to 2.5 mg/kg/day n = 19) without or with topical agents (steroids n = 10; calcineurin inhibitors n = 3). In gastrointestinal manifestations of aGVHD, 9 centers (29%) add topical to systemic steroids, and 3 consider topical steroids as the only treatment for mild gastrointestinal and cutaneous aGVHD. The choice of agent for second-line treatment as well as the sequence of administration are extremely heterogeneous, most likely due to a lack of convincing data published. Most frequently used are mycophenolate mofetil (n = 14) and extracorporeal photopheresis (n = 10). Our survey also demonstrates that clinicians chose salvage therapies for steroid-refractory aGVHD based on their centers'' own clinical experience. [Copyright &y& Elsevier]

Abstract: Objective: Despite important progress in its management, chronic lymphocytic leukemia (CLL) remains incurable with standard therapies. Allogeneic stem cell transplantation (SCT) is a potentially curative therapy for patients with CLL. Polyclonal antithymocyte (or anti-T-cell) globulins (ATGs) are used for conditioning in allogeneic SCT mainly due to their anti-T-cell activity. ATGs however, contain antibodies targeting antigens expressed on various hematopoietic cells including B cells. Methods: We assessed anti-CLL activity of two commercially available ATG preparations at clinically relevant concentrations (10–100μg/ml) in CLL samples from 16 patients. Cytotoxicity was determined by staining with 7-amino-actinomycin D (7-AAD), annexin V and flow cytometry. Results: Both ATG preparations induced marked complement-independent dose-dependent cytotoxicity in all samples. Addition of complement strongly enhanced the cytotoxic effect of both ATG preparations significantly. ATG-induced complement-dependent cytotoxicity (CDC) was at least as high as that observed with Alemtuzumab. Both ATGs enhanced the cytotoxic effect of Fludarabine. Conclusion: ATG is an effective agent against CLL in vitro. We suggest that this potential be taken into consideration when developing stem cell transplantation protocols for patients with CLL. [Copyright &y& Elsevier]

Objective: About one third of patients requiring allogeneic hematopoetic stem cell transplantation (HSCT) would not find a matched sibling or alternative donor. Allogeneic HSCT from matched unrelated and mismatched donors carries an increased risk of graft-vs-host disease (GVHD) and transplant-related mortality (TRM). Materials and Methods: We used anti-thymocyte globulin (ATG-Fresenius) at a median dose of 90 mg/kg body weight as part of a total body irradiation or busulfan-based conditioning regimen for prevention of serious GVHD. All patients received cyclosporine A and short-course methotrexate. We compared outcomes of 65 recipients of human leukocyte antigen (HLA)–mismatched unrelated grafts and 194 recipients of HLA-matched unrelated grafts. Mismatches involved one or two loci. Both groups were comparable in age, graft source, diagnosis, stage of disease, and conditioning regimen, and differed only in dose of ATG administered. Results: For matched and mismatched transplants, respectively, there was no significant difference in graft failure (0.5% vs 3%; p = 0.16), in the cumulative incidence of grade II to IV acute GVHD (45% vs 35%; p = 0.14) and no difference in overall chronic GVHD (42% vs 40%; p = 0.68). Estimated overall survival (OS) and disease-free survival (DFS) at 5 years were 55% vs 50% (p = 0.99) and 47% vs 47% (p = 1.0), respectively. The cumulative incidence of relapse and TRM at 5 years were 24% vs 25% (p = 0.63), and 29% vs 27% (p = 0.59), respectively. Conclusion: Inclusion of ATG-Fresenius in the conditioning regimen permits HSCT from mismatched unrelated donors without excess TRM and GVHD, resulting in identical OS and DFS of recipients of HLA-matched and HLA-mismatched grafts. [Copyright &y& Elsevier]

Abstract: Antithymocyte globulin (ATG) as part of conditioning regimens is known to reduce the incidence and severity of acute and chronic graft-versus-host disease (aGVHD, cGVHD). The influence of ATG on transplant-related mortality (TRM) and disease-free survival (DFS) is controversial, and may depend on the dose and timing of ATG. We retrospectively compared 2 doses of ATG-Fresenius (ATG-F) in patients undergoing matched unrelated donor allogeneic hematopoetic stem cell transplantation (HSCT) for hematologic malignancies. A dose of 60 mg/kg body weight has previously been recommended for ATG-F. All patients received cyclosporine A and short course methotrexate. ATG-F was administered at a dose of 30 mg/kg on day –1 (ATG-30 group, n = 34) or 20 mg/kg/day on days –3 to –1 (ATG-60 group, n = 49). There was no difference in time to leukocyte and platelet engraftment in the 2 groups. The incidence of aGVHD grade II-IV (50% versus 53%, P = .83) and grade III-IV (27 versus 20%, P = .60) was similar in the ATG-30 versus ATG-60 groups, respectively. There was a trend to a higher incidence of cGVHD in the ATG-30 group (59% versus 40%, P = .14). The estimated 3-year incidence of relapse was similar in the ATG-30 and ATG-60 groups (15% versus 16%, P = .84) whereas the 2-year TRM was lower for the ATG-30 group (12% versus 33%, P = 0.02), mainly because of a higher incidence of fatal infections in the ATG-60 group. This resulted in a better DFS (73% versus 51%, P = .07) for the ATG-30 group. ATG-F (30 mg/kg) administered as a single dose on day –1 may lead to better outcome in patients undergoing unrelated donor allogeneic HSCT compared to 60 mg/kg given in 3 equivalent doses. A prospective randomized study comparing these 2 doses of ATG-F is warranted. [Copyright &y& Elsevier]

Objective: Allogeneic stem cell transplantation is a potentially curative therapy for patients with multiple myeloma. Polyclonal antithymocyte globulins (ATG) or monoclonal anti-CD52 (Alemtuzumab) are included in conditioning regimens to enhance engraftment and reduce risk of severe graft-vs-host disease. Because both agents have been reported to induce depletion of B cells, we sought to investigate their cytotoxic activity on myeloma cells. Materials and Methods: Complement-mediated and complement-independent activity of ATG-Fresenius and Alemtuzumab was investigated on four myeloma cell lines (RPMI-8226, U266, KMS-12-BM, and EJM) and bone marrow samples from six myeloma patients. Cytotoxicity was determined by staining with annexin V-fluorescein isothiocyanate and 7-amino-actinomycin D followed by flow cytometry. Results: ATG at a concentration of 500 μg mL−1 induced up to 100% and 85% complement-dependent killing of myeloma cell lines and primary myeloma samples respectively. In the absence of complement ATG still could induce up to 50% and 80% apoptosis in myeloma cell lines and primary myeloma samples, respectively. Preincubation of myeloma cells with a general caspase inhibitor abrogated ATG-induced complement-independent cell death. Alemtuzumab-mediated myeloma cytotoxicity was only observed in KMS-12-BM cells, and in none of the patient samples. Conclusion: ATG induces marked cytotoxic activity both in myeloma cell lines and in primary myeloma samples. Further elucidation of antibodies and antigens involved may pave the way for antibody-based myeloma therapy. [Copyright &y& Elsevier]

• Late relapse after 5 years occurs in about 14% of myelofibrosis patients after allogeneic stem cell transplantation (allo-SCT). • For detection, chimerism and minimal residual disease monitoring should be performed even beyond 5 years after allo-SCT. • Most relapses can be successfully salvaged by donor lymphocyte infusion and/or second allo-SCT. In this cross-sectional study, we retrospectively evaluated the files of 227 patients with myelofibrosis who underwent transplantation between 1994 and 2015 for relapse later than 5 years after allogeneic stem cell transplantation (SCT). A total of 94 patients who were alive and in remission at 5 years were identified with follow-up of at least 5 years (median, 9.15 years) after SCT. Thirteen patients (14%) experienced late molecular (n = 6) or hematologic (n = 7) relapse at a median of 7.1 years while 81 patients did not experience relapse. Relapse patients received either donor lymphocyte infusion (DLI) (n = 7) and/or second transplantation (n = 4). Of those, 72.7% achieved again full donor cell chimerism and molecular remission, and after a median follow-up of 45 months, the 3-year overall survival rates for patients with or without relapse were 90.9% (95% confidence interval [CI], 77% to 100%) and 98.8% (95% CI, 96% to 100%), respectively (P =.13). We conclude that late relapse occurs in about 14% of the patients and the majority can be successfully salvaged with DLI and/or second allograft. All patients with molecular relapse are alive and support the long-time molecular monitoring in myelofibrosis patients after allogeneic SCT. [ABSTRACT FROM AUTHOR]

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation is still associated with significant morbidity and mortality. First-line treatment of cGVHD is based on steroids of 1 mg/kg/day of prednisone. The role of calcineurin inhibitors remains controversial, especially in patients with low risk for mortality (normal platelets counts), whereas patients with low platelets at diagnosis and/or high risk for steroid toxicity may be treated upfront with the combination of prednisone and a calcineurin inhibitor. Additional systemic immunosuppressive agents, like thalidomide, mycophenolic acid, and azathioprine, failed to improve treatment results in the primary treatment of cGVHD and are in part associated with higher morbidity, and in the case of azathioprine, with higher mortality. Despite advances in diagnosis of cGVHD as well as supportive care, half of the patients fail to achieve a long-lasting response to first-line treatment, and infectious morbidity continues to be significant. Therefore, immunomodulatory interventions with low infectious morbidity and mortality such as photopheresis need urgent evaluation in clinical trials. Beside systemic immunosuppression, the use of topical immunosuppressive interventions may improve local response rates and may be used as the only treatment in mild localized organ manifestations of cGVHD. [Copyright &y& Elsevier]

Objective: We investigated the effect of at least two cycles of bortezomib (1.3 mg/m2 intravenously, days 1, 4, 8, and 11) after dose-reduced allogeneic stem cell transplantation (SCT) on toxicity, CD3+ cells, graft-versus-host disease (GvHD), and response in patients with multiple myeloma. Methods: Eighteen patients with multiple myeloma without progressive disease were included. The proteasome inhibitor was given at median of 8 months after allografting to enhance or maintain remission status. Results: Fourteen patients (78%) completed the proposed two cycles. Four patients had to discontinue therapy due to neurotoxicity (n = 3) or gastrointestinal toxicity (n = 1). Severe grade III/IV toxicity was seen for thrombocytopenia (50%), leukopenia (17%), or neuropathy (17%), which was more often seen in patients treated concomitantly with cyclosporine (p = 0.06). The median circulating CD3+ cells decreased during treatment from 550 μL to 438 μL (p = 0.03), resulting in herpes zoster infection in three patients (17%). In three patients, a mild aggravation of existing acute or chronic GvHD of the skin, and in one patient de novo skin grade I acute GvHD was noted. In patients with measurable disease, complete remission, partial remission, and minor response was seen in 3 (30%), 5 (50%), and 2 (20%) patients, respective. Conclusion: Bortezomib after allogeneic SCT is effective but further studies are needed to balance the efficacy with potential hazards such as infectious complications, aggravation of GvHD, and neurotoxicity. [Copyright &y& Elsevier]

Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate. The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing. In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact. The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles. Enveloped hepatitis E virus particles could be identified by PCR and electron microscopy in the ejaculate of immunosuppressed chronically infected patients, but not in immunocompetent experimentally infected pigs or in patients with acute self-limiting hepatitis E. [Display omitted] • HEV genotype 3 particles were found in the ejaculate of patients with chronic HEV. • Genetic variants differed between particles originating from the liver, compared to those originating from the male reproductive system. • HEV shedding in the ejaculate continued for >9 months following the end of viremia. [ABSTRACT FROM AUTHOR]

• Allogeneic stem cell transplantation (SCT) in patients with lower-risk myelodysplastic syndrome (MDS) resulted in very good outcomes. • Transplantation from a matched donor was a significant factor in survival. • The risk of relapse risk is <10% in patients with lower-risk MDS after allogeneic SCT. The indication for allogeneic stem cell transplantation (SCT) in patients with lower-risk myelodysplastic syndrome (MDS) is controversial. Here we report 60 patients with a low risk (n = 32) or intermediate risk (n = 28) classification according to the revised International Prognostic Scoring System (IPSS-R) who underwent allogeneic SCT with a reduced-intensity conditioning (n = 45) or myeloablative conditioning (n = 15) regimen from an HLA-identical sibling (n = 9), a matched unrelated donor (n = 36), or a mismatched unrelated donor (n = 15). The rates of grade II-IV and grade III-IV acute graft-versus-host disease were 32% and 7%, respectively, resulting in a transplantation-related mortality (TRM) of 17% at 3 years. The cumulative incidence of relapse at 5 years was only 7%, resulting in a 5-year disease-free survival of 72% and overall survival (OS) of 79%. Transplantation from a fully matched donor resulted in significantly improved OS at 5 years (91% versus 70%). Allogeneic SCT in lower-risk MDS (IPSS-R low or intermediate risk) from an HLA-matched donor resulted in excellent OS with a low risk of relapse. [ABSTRACT FROM AUTHOR]

• Overall outcomes of patients allografted for BC CML remain poor in the TKI era. • Survival after alloSCT for BC CML depends on the pretransplant remission status. • Conventional prognostic factors remain outcome determinants for BC CML in remission. • UD transplant is associated with better LFS in patients with active BC at alloSCT. The prognosis of patients with blast crisis (BC) chronic myeloid leukemia (CML) is still dismal. Allogeneic stem cell transplantation represents the only curative treatment option, but data on transplant outcomes are scarce. We therefore conducted a retrospective, registry-based study of adult patients allografted for BC CML, focusing on patients with active disease at transplant and pretransplant prognostic factors. One hundred seventy patients allografted for BC CML after tyrosine kinase inhibitor pretreatment between 2004 and 2016 were analyzed. Before transplant, 95 patients were in remission, whereas 75 patients had active BC. In multivariable analysis of the entire cohort, active BC at transplant was the strongest factor associated with decreased overall survival (hazrd ratio, 1.87; P =.010) and shorter leukemia-free survival (LFS; hazard ratio, 1.69; P =.017). For patients with BC in remission at transplant, advanced age (≥45 years), lower performance status (≤80%), longer interval from diagnosis BC to transplant (>12 months), myeloablative conditioning, and unrelated donor (UD) transplant were risk factors for inferior survival. In patients with active BC, only UD transplant was significantly associated with prolonged LFS and trended toward improved overall survival. In summary, survival of patients allografted for BC CML was strongly dependent on pretransplant remission status. In patients with remission of BC, conventional prognostic factors remained the major determinants of outcome, whereas in those with active BC at transplant, UD transplant was associated with prolonged LFS in our study. [ABSTRACT FROM AUTHOR]

• Hepatitis E is associated with liver failure and mortality in haematological malignancy. • Blood-borne transmission contributes to the burden. • Alleviation of immunosuppressive treatment for hepatitis E requires caution. • Ribavirin is effective and should be initiated early. The burden of hepatitis E virus (HEV) infection among patients with haematological malignancy has only been scarcely reported. Therefore, we aimed to describe this burden in patients with haematological malignancies, including those receiving allogeneic haematopoietic stem cell transplantation. We conducted a retrospective, multicentre cohort study across 11 European centres and collected clinical characteristics of 50 patients with haematological malignancy and RNA-positive, clinically overt hepatitis E between April 2014 and March 2017. The primary endpoint was HEV-associated mortality; the secondary endpoint was HEV-associated liver-related morbidity. The most frequent underlying haematological malignancies were aggressive non-Hodgkin lymphoma (NHL) (34%), indolent NHL (iNHL) (24%), and acute leukaemia (36%). Twenty-one (42%) patients had received allogeneic haematopoietic stem cell transplantation (alloHSCT). Death with ongoing hepatitis E occurred in 8 (16%) patients, including 1 patient with iNHL and 1 patient >100 days after alloHSCT in complete remission, and was associated with male sex (p = 0.040), cirrhosis (p = 0.006) and alloHSCT (p = 0.056). Blood-borne transmission of hepatitis E was demonstrated in 5 (10%) patients, and associated with liver-related mortality in 2 patients. Hepatitis E progressed to chronic hepatitis in 17 (34%) patients overall, and in 10 (47.6%) and 6 (50%) alloHSCT and iNHL patients, respectively. Hepatitis E was associated with acute or acute-on-chronic liver failure in 4 (8%) patients with 75% mortality. Ribavirin was administered to 24 (48%) patients, with an HEV clearance rate of 79.2%. Ribavirin treatment was associated with lower mortality (p = 0.037) and by trend with lower rates of chronicity (p = 0.407) when initiated <24 and <12 weeks after diagnosis of hepatitis E, respectively. Immunosuppressive treatment reductions were associated with mortality in 2 patients (28.6%). Hepatitis E is associated with mortality and liver-related morbidity in patients with haematological malignancy. Blood-borne transmission contributes to the burden. Ribavirin should be initiated early, whereas reduction of immunosuppressive treatment requires caution. Little is known about the burden of hepatitis E among patients with haematological malignancy. We conducted a retrospective European cohort study among 50 patients with haematological malignancy, including haematopoietic stem cell transplant recipients, with clinically significant HEV infection and found that hepatitis E is associated with hepatic and extrahepatic mortality, including among patients with indolent disease or among stem cell transplant recipients in complete remission. Hepatitis E virus infection evolved to chronic hepatitis in 5 (45.5%) patients exposed to a rituximab-containing regimen and 10 (47.6%) stem cell transplant recipients. Reducing immunosuppressive therapy because of hepatitis E was associated with mortality, while early ribavirin treatment was safe and effective. [ABSTRACT FROM AUTHOR]

• First-line treatment is applied relatively homogeniously among German, Austrian, and Swiss transplant centers except initial treatment of progressive onset. • Second-line treatment varies considerably with new agents entering clinic through the last 10 years. • Even in the presence of evidence clinical practice in treatment of lung manifestations varies considerably. Chronic graft-versus-host disease (cGVHD) remains the leading cause of late morbidity and mortality. Despite the growing number of treatment options in cGVHD, evidence remains sparse. The German-Austrian-Swiss GVHD Consortium performed a survey on clinical practice in treatment of cGVHD among transplant centers in Germany, Austria, and Switzerland in 2009 and 2018 and compared the results. The survey performed in 2009 contained 20 questions on first-line treatment and related issues and 4 questions on second-line scenarios followed by a survey on all systemic and topic treatment options known and applied, with 31 of 36 transplant centers (86%) responding. The survey in 2018 repeated 7 questions on first-line treatment and 3 questions on second-line scenarios followed by an updated survey on all current systemic treatment options known and applied, with 29 of 66 centers (43%) responding. In summary, the results show a large overlap of first-line treatment practice between centers and the 2 surveys because of a lack of new data that changes practice, except significant heterogeneity of treatment of cGVHD progressive onset type, which can be explained by the lack of trials focusing on this high-risk entity. In contrast, treatment options applied to second-line therapy vary considerably, with new agents like ibrutinib and ruxolitinib entering clinical practice. Moreover, treatment of bronchiolitis obliterans syndrome demonstrates heterogeneity in applied therapeutic options and sequence because of a lack of controlled data and different conclusions from already existing evidence. In summary, the survey results demonstrate an increasing number of treatment options applied to cGVHD accompanied by a significant heterogeneity in second-line treatment and underline the urgent need for clinical trials and registry analyses on rare entities with high mortality like progressive onset type and lung involvement of cGVHD. [ABSTRACT FROM AUTHOR]

Abstract JAK inhibition by ruxolitinib is approved for treating myelofibrosis and also has shown efficacy in treating steroid-resistant acute and chronic graft-versus-host disease (GVHD). In 12 patients with myelofibrosis (median age, 63 years; range, 43 to 71 years) who were treated with ruxolitinib and underwent allogeneic stem cell transplantation (ASCT), ruxolitinib was continued (2 × 5 mg daily) until stable engraftment. No graft failure was observed, and leukocyte engraftment was achieved after a median of 12 days (range, 11 to 18 days). One patient developed fever of unknown origin after discontinuation of ruxolitinib; otherwise, no withdrawal syndrome was observed. Overall, only 1 patient each experienced acute GVHD grade I or II, resulting in an 8% incidence of acute GVHD grade II-IV at day +100, with no nonrelapse mortality. Complete chimerism was achieved in 11 patients after a median of 40 days, and molecular clearance of the underlying driver mutation was noted in 10 patients after a median of 32 days. Cytomegalovirus (CMV) reactivation occurred in 5 patients (41%), 1 of whom had CMV colitis as well, but all resolved after ganciclovir treatment. In 2 patients, ruxolitinib had to be discontinued on day 17 and day 18 after ASCT due to cytopenia after engraftment. Levels of inflammatory cytokines IL-8, IL-10, IL-6, TNFR2, INF-α, and INF-β were reduced after ruxolitinib treatment. After day +100, 4 patients developed acute GVHD (1 with grade I, 2 with grade II, and 1 with grade III) after tapering of cyclosporine, and all patients were alive at a median follow-up of 17 months (range, 12 to 18 months). [ABSTRACT FROM AUTHOR]

The major reason for treatment failure after allografting in multiple myeloma (MM) is relapse. Donor lymphocyte infusions (DLIs) are considered a valuable post-transplant strategy mainly for relapsed patients but using them to prevent relapse in MM has been reported rarely. In the present study, we examined the efficacy of prophylactic DLIs after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in myeloma patients with a long-term follow-up of more than 5 years. A total of 61 patients with MM who did not relapse or develop disease progression after allo-HSCT were treated with prophylactic DLI in an escalating fashion (overall 132 DLI procedures) to deepen remission status and prevent relapse. Overall response rate to DLI was 77%. Thirty-three patients (54%) upgraded their remission status, 41 patients (67%) achieved or maintained complete remission, and 26% achieved a molecular remission. Incidence of acute graft-versus-host disease (GVHD) grade II to IV was 33% and no DLI-related mortality was noted. After a median follow-up of 68.7 months from first DLI the estimated 8-year progression-free survival (PFS), and overall survival (OS) in a landmark analysis was 43% (95% confidence interval [CI], 28% to 57%) and 67% (95% CI, 53% to 82%), respectively, with best outcome for patients who acquired molecular remission (8-year PFS was 62% and 8-year OS was 83%). Prophylactic escalating DLI in a selected cohort of MM patients to prevent relapse after allograft resulted in a low incidence of severe GVHD and encouraging long-term results, especially if molecular remission is achieved. [ABSTRACT FROM AUTHOR]

Background & Aims Routine HEV testing of blood products has recently been implemented in Great Britain and the Netherlands. The relevance of transfusion-transmitted HEV infections is still controversially discussed in Europe. Methods All blood donations at the University Medical Center Hamburg-Eppendorf were prospectively tested for HEV RNA by pooled PCR from October 2016 to May 2017. Reactive samples were individually retested. Additionally, stored samples from previous donations of positive donors were tested to determine the duration of HEV viraemia. HEV RNA-positive donors and a control cohort were asked to answer a questionnaire. Results Twenty-three out of 18,737 HEV RNA-positive donors were identified (0.12%). Only two of the positive donors (8.7%) presented with elevated aminotransferases at time of donation (alanine aminotransferase: 192 and 101 U/L). The retrospective analysis of all positive donors revealed that four asymptomatic donors had been HEV viraemic for up to three months with the longest duration of HEV viraemia exceeding four months. Despite the HEV-testing efforts, 14 HEV RNA-positive blood products were transfused into 12 immunocompromised and two immunocompetent patients. One recipient of these products developed fatal acute-on-chronic liver failure complicated by Pseudomonas septicemia . The questionnaire revealed that HEV RNA-positive donors significantly more often consumed raw pork meat (12 out of 18; 67%) than controls (89 out of 256; 35%; p  = 0.01). In two donors, undercooked pork liver dishes were identified as the source of infection. HEV genotyping was possible in 7 out of 23 of HEV viraemic donors and six out of seven isolates belonged to HEV Genotype 3, Group 2. Conclusions Prolonged HEV viraemia can be detected at a relatively high rate in Northern German blood donors, leading to transfusion-transmitted HEV infections in several patients with the risk of severe and fatal complications. Eating raw pork tartare represented a relevant risk for the acquisition of HEV infection. Lay summary The relevance of transfusion-transmitted hepatitis E virus infections has been discussed controversially. Herein, we present the first report on routine hepatitis E virus screening of blood donations at a tertiary care centre in Germany. Hepatitis E viraemia was found at a relatively high rate of 0.12% among blood donors, which represents a relevant transfusion-related risk for vulnerable patient populations. [ABSTRACT FROM AUTHOR]

Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P  = .05), improved PFS (HR, .393; P  = .01), and OS (HR, .448; P  = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [ P  = .008], and HR, 5.451 [ P  = .002], respectively), whereas no impact was observed for “triple negative” patients. Molecular genetics, especially CALR , IDH2 , and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation remains the sole curative option for myelofibrosis. Many transplantation recipients receive a reduced-intensity conditioning (RIC) regimen owing to age or comorbidities; however, there is little published evidence to guide the choice of RIC regimen. In this study, we compared outcomes in patients who received 1 of 2 frequently used RIC regimens for patients with myelofibrosis: fludarabine-busulfan (FB) and fludarabine-melphalan (FM). A total of 160 patients underwent a RIC allograft procedure (FB group, n = 105; FM group, n = 55). We have developed a complex statistical model involving weighting and adjustment to permit comparison between these 2 groups. After weighting, the incidence of acute graft-versus-host disease (GVHD) was 62% in the FM group and 31% in the FB group ( P = .001), and the corresponding incidence of chronic GVHD was 49% and 53%, respectively. The 7-year progression-free survival was were 52% in the FM group versus 33% in the FB group, and the 7-year overall survival rate 52% in the FM group versus 59% in the FB group. Nonrelapse mortality (NRM) was 43% in the FM group and 31% in the FB group. Multivariable analyses revealed no significant differences in PFS between the 2 groups; however, the relapse rate was significantly lower in the FM group (hazard ratio, 9.21; P = .008), whereas a trend toward reduced NRM was seen in the FB group (hazard ratio, 0.51; P = .068). In conclusion, both regimens appear to be efficient in mediating disease control and can be used to successfully condition patients with myelofibrosis. The FM regimen appears to induce more NRM than the FB regimen, but with augmented control of disease, leading to comparable overall survival rates for both regimens. [ABSTRACT FROM AUTHOR]

Acute graft-versus-host disease (GVHD) remains a leading cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation. The clinical staging of GVHD varies greatly between transplant centers and is frequently not agreed on by independent reviewers. The lack of standardized approaches to handle common sources of discrepancy in GVHD grading likely contributes to why promising GVHD treatments reported from single centers have failed to show benefit in randomized multicenter clinical trials. We developed guidelines through international expert consensus opinion to standardize the diagnosis and clinical staging of GVHD for use in a large international GVHD research consortium. During the first year of use, the guidance followed discussion of complex clinical phenotypes by experienced transplant physicians and data managers. These guidelines increase the uniformity of GVHD symptom capture, which may improve the reproducibility of GVHD clinical trials after further prospective validation. [ABSTRACT FROM AUTHOR]

We correlate regression of bone marrow fibrosis (BMF) on day 30 and 100 after dose- reduced allogeneic stem cell transplantation (allo-SCT) in 57 patients with primary or post–essential thrombocythemia/polycythemia vera myelofibrosis with graft function and survival. The distribution of International Prognostic Scoring System (IPSS) risk score categories was 1 patient with low risk, 5 patients with intermediate-1 risk, 18 patients with intermediate-2 risk, and 33 patients with high risk. Before allo-SCT, 41 patients (72%) were classified as XXX [myclofibrosis (MF)]-3 and 16 (28%) were classified as MF-2 according to the World Health Organization criteria. At postengraftment day +30 (±10 days), 21% of the patients had near-complete or complete regression of BMF (MF-0/-1), and on day +100 (±20 days), 54% were MF-0/-1. The 5-year overall survival rate at day +100 was 96% in patients with MF-0/-1 and 57% for those with MF-2/-3 (P = .04). There was no difference in BMF regression at day +100 between IPSS high-risk and low/intermediate-risk patients. Complete donor cell chimerism at day +100 was seen in 81% of patients with MF-0/-1 and in 31% of those with MF-2/-3. Patients with MF-2/-3 at day +100 were more likely to be transfusion-dependent for either RBCs (P = .014) or platelets (P = .018). Rapid BMF regression after reduced-intensity conditioning allo-SCT resulted in a favorable survival independent of IPSS risk score at transplantation. [ABSTRACT FROM AUTHOR]

Abstract: We retrospectively analyzed outcomes of a CD34+-selected stem cell boost (SCB) without prior conditioning in 32 patients (male/22; median age of 54 years; range, 20 to 69) with poor graft function, defined as neutrophils ≤1.5 x 109/L, and/or platelets ≤30 x 109/L, and/or hemoglobin ≤8.5 g/dL). The median interval between stem cell transplantation and SCB was 5 months (range, 2 to 228). The median number of CD34+ and CD3+ cells were 3.4 x 106/kg (.96 to 8.30) and 9 x 103/kg body weight (range, 2 to 70), respectively. Hematological improvement was observed in 81% of patients and noted after a median of 30 days (range, 14 to 120) after SCB. The recipients of related grafts responded faster than recipients of unrelated grafts (20 versus 30 days, P = .04). The cumulative incidence of acute (grade II to IV) and chronic graft-versus-host disease (GVHD) after SCB was 17% and 26%, respectively. Patients with acute GVHD received a higher median CD3+ cell dose. The 2-year probability of overall survival was 45%. We suggest that SCB represents an effective approach to improve poor graft function post transplantation, but optimal timing of SCB administration, anti-infective, and GVHD prophylaxis needs further evaluation. [Copyright &y& Elsevier]

Abstract: Within a prospective protocol, the incidence and impact of achievement of molecular remission (mCR) and high-risk cytogenetics was investigated in 73 patients with multiple myeloma (MM) after autologous (auto)–allogeneic (allo) tandem stem cell transplantation (SCT). After induction chemotherapy, patients received melphalan 200 mg/m2 before undergoing auto-SCT, followed 3 months later by melphalan 140 mg/m2 and fludarabine 180 mg/m2 before allo-SCT. Sixteen patients had high-risk cytogenetic features, defined by positive FISH for del(17p13) and/or t(4;14). Overall, 66% of the patients achieved CR or near-CR, and 41% achieved mCR, which was sustained negative (at least 4 consecutive samples negative) in 15 patients (21%), with no significant difference in incidence between the patients with high-risk cytogenetics and others (P = .70). After a median follow-up of 6 years, overall 5-year progression-free survival was 29%, with no significant difference between del 17p13/t(4;14)-harboring patients and others (24% versus 30%; P = .70). The 5-year progression-free survival differed substantially according to the achieved remission: 17% for partial remission, 41% for CR, 57% for mCR, and 85% for sustained mCR. These results suggest that auto–allo tandem SCT may overcome the negative prognostic effect of del(17p13) and/or t(4;14) and that achievement of molecular remission resulted in long-term freedom from disease. [Copyright &y& Elsevier]

Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8+ T cell activity. We investigated an automated clinical-scale human-CD4+-cell purification method to deplete CD8+ cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4+ cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS®) before DLIs. MACS resulted in a mean CD4+ cell count of 16 × 106/kg bw corresponding to 3.4-fold CD4+ cell enrichment. Mean yield and purity of CD45+CD3+CD4+CD14−7AAD− were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 106 CD4+/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4+ by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD. [Copyright &y& Elsevier]

Steroid refractory chronic graft-versus-host disease (cGVHD) is associated with a significant morbidity and mortality. Although first-line treatment of cGVHD is based on controlled trials, second-line treatment is almost solely based on phase II trials or retrospective analyses. The consensus conference on clinical practice in cGVHD held in Regensburg aimed to achieve a consensus on the current evidence of treatment options as well as to provide guidelines for daily clinical practice. Treatment modalities are the use of steroids and calcineurin inhibitors as well as immunomodulating modalities (photopheresis, mTOR-inhibitors, thalidomide, hydroxychloroquine, vitamin A analogs, clofazimine), and cytostatic agents (mycophenolate mofetil, methotrexate, cyclophosphamide, pentostatin). Recent reports showed some efficacy of rituximab, alemtuzumab, and etanercept in selected patients. Moreover, tyrosine kinase inihibitors such as imatinib came into the field because of their ability to interfere with the platelet-derived growth factor (PDGF-R) pathway involved in fibrosis. An other treatment option is low-dose thoracoabdominal irradiation. Although different treatment options are available, the “trial-and-error system” remains the only way to identify the drug effective in the individual patient, and valid biomarkers are eagerly needed to identify the likelihood of response to a drug in advance. Moreover, the sparse evidence for most treatment entities indicates the urgent need for systematic evaluation of second-line treatment options in cGVHD. [ABSTRACT FROM AUTHOR]

Objective: Chimerism is well-established for surveillance of acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (HSCT), but interpretation of the results and techniques is not standardized. Materials and Methods: We correlated chimerism in 75 AML patients (38 male, 37 female) who underwent myeloablative (n = 36)/reduced (n = 39) allo-HSCT with the risk of relapse and survival. Chimerism was evaluated by quantitative real-time polymerase chain reaction for donor/recipient specific polymorphisms/Y-specific sequences. Results: After HSCT, 40 patients (53%) achieved stable complete donor chimerism (≥99.0% of donor alleles), while 35 (47%) failed to achieve stable donor chimerism. Thirty-one patients (41%) showed decreasing donor alleles after having first achieved complete donor chimerism. To investigate the kinetics of mixed chimerism, patients were separated whether they showed subsequent increasing or decreasing donor alleles. Subsequent decrease of donor alleles was associated with relapses in 17 of 18 cases (94%), while no patient with subsequent increasing donor alleles relapsed (p < 0.001). Patients with mixed chimerism and increasing donor alleles had better 2-year disease-free survival (85%) than those with decreasing donor alleles (0%; p < 0.001). Conclusions: The kinetics of mixed chimerism as assessed by quantitative real-time polymerase chain reaction is an important prognostic predictor in the post-transplantation period of AML patients. [ABSTRACT FROM AUTHOR]

Objective: To investigate post-transplant immunotherapy with escalating donor-lymphocyte infusions (DLI) and novel agents (thalidomide, bortezomib, and lenalidomide) to target complete remission (CR). Materials and Methods: Thirty-two patients with multiple myeloma who achieved only partial remission after allogeneic stem cell transplantation were treated with DLI. If no CR was achieved, one of the novel agents was added to target CR. Results: CR defined either by European Group for Blood and Marrow Transplantation criteria, flow cytometry, or molecular methods as assessed by patient-specific immunoglobulin H–polymerase chain reaction or plasma cell chimerism polymerase chain reaction was accomplished in 59%, 63%, and 50% of patients, respectively. Achievement of CR resulted in improved 5-year progressive-free and overall survival, according to European Group for Blood and Marrow Transplantation criteria (53% vs 35%; p =0.03 and 90% vs 62%; p =0.06), flow cytometry (74% vs 15%; p =0.001 and 100% vs 52%; p =0.1), or molecular methods (84% vs 38%; p =0.001 and 100% vs 71%; p =0.03). Conclusions: Our finding demonstrates the clinical relevance of posttransplantation therapies to upgrade remission, and of remission''s depth for long-term survival in myeloma patients. [Copyright &y& Elsevier]

Abstract: Between August 1996 and December 2004, 369 patients with a median age of 41 years (range: 1-68 years) received stem cell transplantation (SCT) from unrelated donors after an antithymocyte-globulin (ATG)-containing conditioning regimen. In 268 patients, complete molecular typing (4-digit) of HLA-A, -B, -C, -DRB1, and -DQB1 was available: 110 patients were completely matched for 10 alleles, 91 patients had 1 allele-mismatch (9/10), and 67 patients were mismatched for 2-4 alleles (6-8/10). The incidence of grade II-IV acute graft-versus-host disease (aGVHD) was 33% in the 10/10, 41% in the 9/10, and 40% in the 6-8/10 group, respectively (P = .1). The cumulative incidence of treatment-related mortality (TRM) and relapse among the groups were similar (27%, 31%, and 32%, P = .2; and 28%, 27%, and 26%, P = .9. After a median follow-up of 35 months (range: 3-120 months), the estimated 5-year disease-free survival (DFS) was 42% and did not differ among the 10/10, the 9/10, and the 6-8/10-mismatched groups (45% versus 42% versus 39%) (P = .5). In multivariate analysis, only age (hazard ratio [HR] 1.013) (P = .004) and bad-risk disease (HR 1.975) (P < .001) were independent risk factors for DFS. In conclusion, pretransplant ATG allows allogeneic SCT from unrelated donors with HLA disparities. [Copyright &y& Elsevier]

Objective: To investigate lineage-specific chimerism of plasma cells after allogeneic transplantation by real-time PCR based on bi-allelic sequence polymorphism or, in case of female-to-male transplantation, on the detection of the DFFRY gene and to determine its value to quantify minimal residual disease in myeloma patients. Methods: Forty-eight samples from bone marrow samples and peripheral blood from 34 nonmyeloma patients were analyzed at different times after transplantation. Sixty-two samples from 22 myeloma patients were analyzed at different times after allogeneic stem cell transplantation, and results were compared with immunofixation and, in some cases, with PCR data using patient-specific primers. Results: The median chimerism for T cells at day +100 was greater than 99.9% and remained stable on day +180 and 1 year after transplantation. In contrast, the median donor plasma cell chimerism at day +100 was 95.5%, at day +180 98.6%, at day +360 99.8%, and 2 or more years after transplantation greater than 99.9%. Sensitivity of real-time PCR using human short insertion/deletion polymorphisms (SIDP) was 10-4 and in case of Y-PCR 10-5. Sequential monitoring of donor plasma cell chimerism showed that increasing and stable chimerism were associated with ongoing remission in 15 out of 16 samples (93%), and decreases in chimerism predicted relapse in 5 out of 6 patients. Conclusion: We conclude that plasma cell chimerism after allogeneic stem cell transplantation is delayed in comparison to T-cell chimerism. Sequential quantitative measurement of plasma cells after allogeneic stem cell transplantation with highly sensitive real-time PCR allows monitoring of residual host-tumor cells in patients with multiple myeloma and allows guiding adoptive immunotherapy strategies to enhance remission status and to prevent clinical relapse. [Copyright &y& Elsevier]

We evaluated prognostic factors of melphalan/fludarabine-based dose-reduced allografts in patients with multiple myeloma. From 1998 to 2002, 120 patients with multiple myeloma were treated with melphalan/fludarabine followed by allogeneic stem cell transplantation. The cumulative risk at 1 year for treatment-related mortality (TRM) was 18% (95% confidence interval [CI], 12%–28%). In a multivariate analysis, relapse after prior high-dose chemotherapy was the most significant risk factor for TRM (hazard ratio [HR], 2.80; 95% CI, 1.16–6.74; P = .02), relapse (HR, 4.14; 95% CI, 2.04–8.38; P < .001), event-free survival (HR, 3.11; 95% CI, 1.77–5.46; P < .001), and overall survival (HR, 2.69; 95% CI, 1.35–5.35; P = .005). In addition, relapse was also significantly diminished by chronic graft-versus-host disease (GVHD) in a time-dependent Cox model (HR, 0.37; 95% CI, 0.16–0.87; P = .02). At transplantation, 8% of the patients were in complete remission, whereas 27% had progressive disease. After allografting, 49% achieved complete remission, and 38% achieved partial remission. In a subgroup of patients with chemosensitivity at transplantation and no relapse after prior high-dose chemotherapy who underwent transplantation with peripheral blood stem cells (n = 46), the cumulative risk of TRM at 1 year was only 8% (95% CI, 1%–54%). The 2-year estimated event-free and overall survival was 60% (95% CI, 42%–78%) and 75% (95% CI, 59%–91%), respectively, for related donors (n = 34) and was 81% (95% CI, 59%–100%) and 92% (95% CI, 76%–100%), respectively, for unrelated donors (n = 12). [Copyright &y& Elsevier]

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2×109) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34+ and AC133+ bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. [Copyright &y& Elsevier]

Acute GVHD biomarkers predict long-term outcomes after GVHD diagnosis. Our group has validated the MAGIC algorithm which uses the concentrations of 2 GVHD biomarkers, ST2+REG3a, to predict lethal GVHD (defined as death from GVHD without relapse) [Hartwell 2017]. Other published biomarker combinations that predict GVHD outcomes include ST2+ REG3a+TNFR1 [Levine 2015], ST2+TIM3 [Abu Zaid 2017], ST2+TNFR1 [McDonald 2015], TIM3+TNFR1+IL6 [McDonald 2015], as well as AREG alone [Holtan 2018]. It is not clear which biomarker combination best predicts lethal GVHD because these algorithms were developed using different patient cohorts with different endpoints. To answer this question, we compared the predictive accuracy of different biomarker combinations as well as these six specific combinations in the same patient cohort. We studied 522 patients with serum samples at GVHD diagnosis who were transplanted at 19 Mount Sinai Acute GVHD International Consortium (MAGIC) centers between January 1, 2004 and April 30, 2017. Patients were divided into training (n = 253) and validation (n = 269) sets; validation patients were transplanted after November 1, 2015 and had not previously been used to generate an algorithm. Biomarkers were measured by ELISA and log-transformed values were used to predict 1-year lethal GVHD by competing risk regression. Four of the 6 biomarkers (ST2, REG3a, TNFR1, and TIM3) independently predicted lethal GVHD in the training set. We then developed algorithms that predicted lethal GVHD using all possible combinations of these 4 biomarkers as well as the published combination of TIM3+TNFR1+IL6 and AREG alone. The best algorithms of 1-4 biomarkers for predicting lethal GVHD were REG3a alone, ST2+REG3a, ST2+REG3a+TNFR1, and ST2+REG3a+TNFR1+TIM3. While ST2+REG3a was the most accurate combination based on the lowest Akaike Information Criterion (AIC), the other published biomarker combinations produced similar AICs. Therefore, we used the independent validation set to compare all six published algorithms. We generated area under the receiver operating characteristic curves (AUC) for each algorithm (FIG 1). We next determined the threshold that maximized sensitivity and specificity for each algorithm and calculated the cumulative incidence of lethal GVHD in the resulting high and low risk strata (FIG 2). Highly similar results were obtained when the cumulative incidence of non-relapse mortality was used as the endpoint. In a validation set of previously unanalyzed patients, several biomarker combinations reproducibly stratified patients with GVHD for risk of death. The best 4 algorithms, all of which included ST2, produced comparable outcomes. Adding TNFR1 to ST2+ REG3a did not improve accuracy. The ST2+REG3a algorithm best identifies patients at onset of GVHD for high risk of lethal GVHD and NRM, and thus remains a standard for biomarker-based risk prediction. [ABSTRACT FROM AUTHOR]

There are no validated biomarkers that measure a patient's response to therapy for acute graft-versus-host disease (GVHD), the leading cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplant (HCT). Recent studies from the Mount Sinai Acute GVHD International Consortium (MAGIC) have validated an algorithm probability (MAP) that combines serum concentrations of two biomarkers of GVHD (REG3α and ST2) to generate an estimated probability of 6 month NRM for individual patients. The MAP estimates GVHD-mediated damage to crypts throughout the lower GI tract at single time points (Hartwell et al., JCI Insight , 2017; Major- Monfried et al., Blood , 2018). We hypothesized that the change in MAP between start of treatment and 28 days later could serve as a response biomarker and would compare favorably to clinical response, the gold standard which is widely used as a surrogate for long term survival and is the primary endpoint in most GVHD treatment trials (Martin et al., BBMT , 2009; MacMillan et al., Blood , 2010). We prospectively collected serum samples and clinical staging from 368 sequential HCT patients who received systemic treatment for acute GVHD in one of 20 MAGIC centers between January 2016 and February 2018. We computed MAPs and clinical responses for each patient. MAPs of patients who experienced 6 month NRM increased significantly compared to MAPs of patients who survived (p=0.0004). In patients whose initial MAPs were low (Ann Arbor 1, MAP < 0.141) or intermediate (Ann Arbor 2, 0.141 ≤ MAP ≤ 0.290), 6 month NRM clustered among those with the greatest increases in MAP after 28 days (Fig 1A,B). In patients with high initial MAPs (Ann Arbor 3, MAP > 0.290), those who survived tended to have the largest decreases in MAP (Fig 1C). We found that patients whose MAPs rose above the previously determined high-risk threshold MAP of 0.290 had significantly worse survival compared to those who remained below it, whereas the large number patients with initially high MAPs that remained above the threshold had a large increase in mortality (Fig 2). When measured at day 28, MAPs predicted NRM more accurately than clinical responses, with areas under the receiver operating characteristic curve (AUC) of 0.86 and 0.70, respectively (p<0.0001). An algorithm that combined clinical response with biomarkers generated the same AUC as the MAP alone (0.83 v 0.86, p = NS). The same MAP threshold predicted risk of NRM within both clinical responders and non-responders after four weeks of treatment (Fig 3), and in those with and without significant lower GI symptoms (Fig 4). We conclude that the MAP is, to our knowledge, the first laboratory test validated as a response biomarker for acute GVHD treatment and more accurately predicts survival than clinical response after 28 days of treatment. The MAP may serve as a novel endpoint in future trials of GVHD treatment. [ABSTRACT FROM AUTHOR]

Lenalidomide may prevent relapses after allogeneic stem cell transplantation by promoting the immune-mediated graft-versus-tumor effect. We performed a prospective phase I/II study to define the dose-limiting toxicity and the immunologic effects of lenalidomide given early (day 100–180) after allograft for four cycles in patients with multiple myeloma. According to the Fibonacci design, 24 patients with a median age of 53 years were included. Dose-limiting toxicity was organ toxicity owing to graft-versus-host disease, and the maximum tolerable dose was 5 mg. The incidence of graft-versus-host disease after lenalidomide was 38%, occurring after a median of 22 days, and was beside organ toxicity, a leading cause to discontinue the study in 29% of the patients. Immune monitoring revealed a significant increase in peripheral γ-interferon–secreting CD4+ and CD8+ T cells within the first week of lenalidomide treatment followed by a delayed increase in T regulatory cells. Furthermore, natural killer (NK) cells isolated from the peripheral blood of patients evidenced a significantly improved antimyeloma activity after lenalidomide treatment. The immune effect might have contributed to the increased CR rate from 24–42% after lenalidomide treatment because nonresponding patients showed significantly less natural killer and T cell activation. (Study registered under: NCT 00778752.) [ABSTRACT FROM AUTHOR]