Impact of Non-CMV Specific Intravenous Immunoglobulin on Intravenous Ganciclovir Effectiveness

Abstract Introduction Cytomegalovirus (CMV) reactivation is one of the main problems in allogenic hematopoietic stem cell transplantation (allo-HSCT). Standard of care for CMV-disease is still ganciclovir at 10 mg/kg b.i.d. Use of non-CMV specific intravenous immunoglobulin (IVIG) is still controversial and mostly determined by internal care protocols in every transplant center. Here we report data about impact of non-CMV specific IVIG during ganciclovir treatment in allo-HSCT CMV-patients. Patients and methods Thirty two patients with hematological malignancies transplanted in NRCH were included in this study. Detailed patients characteristics are listed in Table 1. All patients were CMV-positive by PCR (Amplisens EBV/ CMV /HHV6-screen-FL", InterLabService, Russia) and required to start therapy with ganciclovir 10 mg/kg when was included to this study. Patients in IVIG group also received non-CMV specific IVIG during ganciclovir treatment with a median total dose 20 g (7.5-90 g.). Kaplan-Meier estimator was used to determine probability of achievement of CMV-negativity from time of ganciclovir was started to CMV-negativity (by PCR) or last contact. The logrank test is used to compare differences between two groups. Fisher's exact test was used for 2×2 tables. P-value less than 0.05 was considered statistically significant Results Influence of IVIG in allo-HSCT patients on probability of achievement of CMV-negativity (by PCR) are shown in the Figure 1. As we can see there is no significant differences on the achievement of CMV-negativity (by PCR) in patients with/without IVIG during ganciclovir therapy. Conclusion The use of non-CMV specific immunoglobulins after allo-HSCT still lies in the plane of faith and the possibilities of the transplantation center than any evidence base. Using IVIG in patients after allo-HSCT is still not a routine in terms of CMV-reactivation/disease therapy and mostly based on an "experience" of every transplant center. According to our data there is no difference between IVIG/no-IVIG groups during ganciclovir treatment so use of IVIG, for our opinion, does not really make sense for CMV-disease treatment. Anyway a large randomized trial is required for determine indications for therapy with IVIG. Disclosures No relevant conflicts of interest to declare.

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"Low Dose Ganciclovir for Cytomegalovirus Reactivation after Allogeneic HSCT Is a Feasible Option with Same Efficacy and Less Toxicity Than Regular Dose"

Blood ◽

10.1182/blood-2020-142112 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Roberta Azevedo ◽

Vinicius Campos Molla ◽

Jessica Fernandes Ramos ◽

Danilo Belchior Ponciano ◽

Pedro Henrique Arruda De Moraes ◽

...

Keyword(s):

Low Dose ◽

Conflicts Of Interest ◽

Prospective Trial ◽

Underlying Disease ◽

Preemptive Therapy ◽

Hematologic Toxicity ◽

Outpatient Basis ◽

Hematopoietic Stem ◽

Cmv Disease ◽

Cmv Reactivation

Background: Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (HSCT) occurs in approximately 70% of seropostive patients. CMV reactivation is associated with higher non-relapse mortality (NRM) and worse overall survival (OS). Although preemptive therapy effectively prevents CMV disease, toxicity of ganciclovir (GCV), valganciclovir (VGCV), and foscarnet is a major concern. While GCV 5 mg/kg BID (regular dose) is the most commonly used regimen, some series have suggested that a lower dose (5mg/kg QD, lower dose) could be an option. Objectives: To compare two different regimens of ganciclovir (regular vs. lower dose) for preemptive CMV therapy. Patients and Methods: This was an observational, retrospective study in adult patients who underwent HSCT between April 2007 and April 2020 in two centers. Doses of ganciclovir were determined at the discretion of the transplant physician. The primary endpoint was CMV clearance (rate and time to) between the two preemptive strategies. Results: We analyzed 118 consecutive patients. The median age was 50 years, acute leukemia was the most frequent underlying disease (59%), and most patients received transplants from alternative donors (matched unrelated in 32% and haploidentical in 36%), after reduced-intensity conditioning regimens (76%), with peripheral blood as the source of stem cells (73%). T-cell depletion was performed in 31% (ATG in 29%, alemtuzumab in 2%). In total there were 174 CMV reactivations: 124 (71%) were treated with the regular dose, and 50 (29%) in an outpatient setting with low dose. The median time to CMV clearance was similar between regular and low dose of GCV (15 days vs. 18 days, respectively, p=0.88). The cumulative incidence (CI) of CMV clearance at 30 days was 83% in the regular dose and 88% in the lower dose (p=0.82). By multivariate analysis, correcting for the differences between the groups, the GCV regimen did not influence the time to CMV clearance (hazard ratio 0.94, 95% confidence interval 0.70 - 1.26). On the other hand, hematologic toxicity was more frequent in the regular dose, with more cases of both grade 3-4 neutropenia (59% vs. 33%, respectively, p=0.002) and thrombocytopenia (77% vs. 48%, respectively, p<0.0001). Ten patients had CMV disease and were treated with GCV 5mg/kg BID for 21 days. Conclusion: Our findings suggest that the use of GCV once daily was safe, less toxic, and may be less expensive, considering that most patients will receive the regimen in an outpatient basis. These data should be confirmed in a prospective trial. Disclosures No relevant conflicts of interest to declare.

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Oral Valganciclovir As Preemptive Therapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Blood ◽

10.1182/blood.v124.21.5855.5855 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5855-5855 ◽

Cited By ~ 1

Author(s):

Vildan Ozkocaman ◽

Fahir Ozkalemkas ◽

Ridvan Ali ◽

Yasemin Karacan ◽

Tuba Ersal ◽

...

Keyword(s):

Stem Cell ◽

Viral Load ◽

Myeloid Leukemia ◽

Preemptive Therapy ◽

Cell Transplant ◽

Cmv Infection ◽

Intravenous Ganciclovir ◽

Hematopoietic Stem ◽

Cmv Disease ◽

Cmv Reactivation

Abstract Introduction: Cytomegalovirus (CMV) infection remains one of the most important complications after allogeneic hematopoietic stem cell transplantation (AlloHSCT). Preemptive therapy with oral VGC or intravenous ganciclovir has replaced universal prophylaxis. We investigated the effect of oral VGC as preemptive therapy on CMV reactivation or infection in AlloHSCT. Patient & Methods: We retrospectively studied 30 consecutive adult recipients of HLA-identical sibling allogeneic peripheral blood stem cell transplant from June 2011 through June 2014 to analyze the safety and efficacy of preemptive therapy for the treatment of CMV infection. Among 30 patients we studied the results of 13 patients with CMV DNA positivity. All 13 recipients and their donors were seropositive for CMV Ig G status. Their diagnosis of pretransplantation were as 8 acute myeloid leukemia (61 %) and 5 acute lymphocytic leukemia (39 %). Median age was 39 years (range 21-54). There were 9 men and 4 women (Table-1). The patients received no prophylaxis against CMV. Blood/serum samples from all patients were routinely monitored for CMV-DNA using PCR twice weekly. We iniciated pre-emptive therapy for CMV as the viral load was above and equal to 500 copies/ml in two consecutive samples. VGC was given twice daily 900 mg for 2 weeks, and maintened at a dose daily 900 mg until viral load is undetectable. Results: The PCR test was persistently negative in 17 patients (57 %) and positive at least once in 13 (43%)patients. All patients with CMV reactivations were on immunsupressive treatment. CMV infection was cleared in most patients within 2 weeks, but for those with stable or increasing CMV levels, the induction period was continued. Twelve patients were treated with oral VGC on an outpatient basis and they all became CMV negative after the first week of treatment. Only one patient received intravenous ganciclovir and he was also CMV negative after the first week of treatment. While no positivity was identified in any of the patients who received VGC on day 21, clinically unimportant low titer CMV positivity was noted in three of these patients. VGC was continued at same dose and no positivity was detected after 2-3 weeks. We were obligated interrupt VGC in only four patients for serious side effects namely neutropenia and thrombocytopenia. We did not observe CMV-related mortality. Conclusions: We concluded that VGC therapy could be safely used at outpatient clinics, with frequent monitorization to prevent severe myelotoxicity. In conclusion, based on our findings, oral VGC is effective and safe in the pre-emptive treatment of CMV disease following allo-HSCT. Therefore, preemptive strategy by oral VGC appears preferable to the prevention of CMV disease in alloHSCT with its advantage of effectiveness and usage in outpatient clinic condition. Table- 1: Management and outcome of the patient characteristics with CMV-reactivation Patients with CMV reactivation/Total number of patient 13/30 Median age, years (range) 39 (21-54) Gender Male Female 9 4 Diagnosis AML ALL 8 5 CMV serologic status Donor+/Recipient + 13 Preperative regimen Bu-Cy CY-TBI Flu/Amsc(FLAMSA) 8 4 1 GVHD propylaxis CSA+MTX CSA+MTX+MM 12 1 GVHD prior to CMV reactivation Acute chronic without GVHD 7 1 5 Prednisolone treatment at the time of starting VCG Yes No 8 5 IST at the time of starting VGC Yes No 13 0 Median duration of CMV reactivation (day) 44 (8-330) Viral load before antivial treament (copies/ml) 1153 (78-12800) Treatment VGC (900 mg, twice daily for induction) GC (5 mg/kg, twice daily for induction) VGC+GC 9 (70 %) 1 (7 %) 3 (23 %) Total treatment duration (day) 24 (10-51) CMV DNA at the end of the treatment Conversion to negative Persistently positive (low titer < 50 copies/ml) 10 (77 %) 3 (23 %) Serious side effects Interruption of therapy due to neutropenia Interruption of therapy due to thrombocytopenia Reactivation 3 (23 %) 1 (7 %) 2 (15 %) Abbreviations: AML: acute myeloid leukemia, ALL: acute lymphoblastic leukemia, CMV: cytomegalovirus,GVHD: graft-versus host disease, CSA: cylosporine, MTX: methotrexate, MMF: mycophenolate mofetil, PDN:prednizolone, IST: Immunosupressive treatment, VGC: valganciclovir, GC: ganciclovir, Bu: Busulfan, Cy: cylophosphamide, TBI: total body irradiation Disclosures No relevant conflicts of interest to declare.

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Evaluation of Cytomegalovirus (CMV)-Specific Cytotoxic T Lymphocyte (CTL) Monitoring Procedures with Tetramer and Intracellular IFN-γ Assay after Allogeneic Hematopoietic Stem Cell Transplantation (SCT).

Blood ◽

10.1182/blood.v104.11.3161.3161 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3161-3161

Author(s):

Yuriko Morita ◽

Mami Hosokawa ◽

Yuji Heike ◽

Tohru Sasaki ◽

Michiko Ebisawa ◽

...

Keyword(s):

T Lymphocyte ◽

Cytotoxic T Lymphocyte ◽

Disease Onset ◽

Hematopoietic Stem ◽

Ifn Γ ◽

Cmv Colitis ◽

Cmv Disease ◽

Cmv Reactivation ◽

Matched Donor ◽

Cmv Antigenemia

Abstract Previous studies on the tetramer-based quantification of CMV-specific CTL have shown that the reconstitution of CMV-specific CTL to levels greater than 10/μL may protect against CMV reactivation. To evaluate the usefulness of CMV-specific tetramer for monitoring the number of CMV-specific CTL, assays with CMVpp65 495–503 tetramer were performed in 34 patients with HLA-A02 while CMVpp65 328–336 tetramer was used in 47 HLA-A24 patients who received non-T-cell-depleted SCT from a serologically full-matched donor after a myeloablative or nonmyeloablative regimen without ATG. Patients were assessed after recovery from CMV reactivation. Although the average number of CTL detected in patients with HLA-A02 [23.5/μL (1.33%/lymphocytes)] was significantly higher than that in patients with HLA-A24 [0.48/μL (0.02%)], the CMV reactivation rate was similar (67.6% and 62.5% in HLA-A02 and A24, respectively). This result demonstrates that these assays are of limited value since the number of CTL detected varies among different HLA-restricted epitopes. To further evaluate whether there is any relationship between CMV-specific CTL and CMV reactivation, the number of CTL in HLA-A02 patients was assessed in detail. The average number of CTLs in 11 patients without CMV reactivation, 23 with CMV reactivation, 13 with a peak CMV antigenemia of >10/50000, and 3 who developed CMV disease was 12.3/μL (0.85%), 29.3/μL (1.35%), 13.8/μL (0.8%) and 16.3/μL (1.33%), respectively. No significant correlation was observed between the number of CTL and CMV reactivation after the reconstitution of CMV immunity. Since CMV reactivation usually occurs within 100 days after SCT, tetramer was assessed biweekly until day 100 in 13 HLA-A02 patients. In those who had CMV reactivation, simultaneous intracellular IFN-γ staining was performed with the same peptide used for tetramer. CMV reactivation was observed in 10 patients between day 23 and day 56 (median, day 34); among them, 5 had a peak antigenemia of >10/50000, and required GCV therapy, and 3 developed CMV colitis. The average number of CTL at CMV reactivation was 5.67 (0.08–22.65) /μL in 10 patients who had reactivation, with 3 showing >10/μL, while this was 1.08 (0–1.98) /μL at day 30 in those who did not. Two of the 3 patients who developed CMV colitis had >10/μL CTL at the time of disease onset, while among 8 who did not require GCV therapy, only 1 and 2 patients recovered CTL >10/μL at day 30 and day 60, respectively. The number of intracellular IFN-γ-secreting cells among those with CMV colitis was 18.2/μL (1.8%) at the time of disease onset, and this increased to 47.3/μL (3.5%) after recovery from CMV disease. These results suggest that tetramer-based monitoring of CTL is of limited value in predicting CMV reactivation compared to intracellular IFN-γ assay that assesses the functional properties of CTL.

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Pre-Transplant Ganciclovir and High-Dose Valacyclovir Prophylaxis Decrease Incidence of CMV Reactivation In High-Risk Seropositive UCBT Recipients

Blood ◽

10.1182/blood.v116.21.678.678 ◽

2010 ◽

Vol 116 (21) ◽

pp. 678-678

Author(s):

Filippo Milano ◽

Steven Pergam ◽

Hu Xie ◽

Jonathan Gutman ◽

Ivy Riffkin ◽

...

Keyword(s):

High Risk ◽

Proportional Hazards Model ◽

Conflicts Of Interest ◽

Standard Dose ◽

Cox Proportional Hazards Model ◽

Prevention Strategy ◽

High Dose ◽

Hematologic Toxicity ◽

Cmv Disease ◽

Cmv Reactivation

Abstract Abstract 678 Background: Umbilical cord blood transplant (UCBT) recipients are high risk for cytomegalovirus (CMV) complications due to delayed and insufficient immune reconstitution. Since CMV viral load has been shown to be associated with the development of disease, an intensified prevention strategy was adopted at the FHCRC (Seattle, WA) which consists of pre-transplant ganciclovir (from day -8 to day -2), and high-dose acyclovir ([HDA] 2 gm valacyclovir 3 times daily) with preemptive bi-weekly monitoring for CMV DNA in serum from day 0 until day +100. Methods: We set out to compare rates of CMV reactivation and disease through day +100 in high-risk CMV seropositive UCBT recipients who received either the intensified strategy (G+HDA) or standard dose of acyclovir/valacyclovir (SDA, acyclovir 800 mg or valacyclovir 500 mg twice daily). All patients underwent weekly plasma testing for CMV by polymerase chain reaction (PCR). Our primary outcomes of interest were any CMV reactivation or disease by day 100. Risk factors for CMV reactivation were assessed using a multivariate Cox proportional hazards model. Results: Of the 105 UCBT recipients transplanted at the FHCRC between 1/2006 and 12/2009, 61 (58%) were CMV seropositive and eligible for inclusion in the cohort. In total, 31/61 (51%) received SDA and 30 (49%) G+HDA. The median patient age was lower in the SDA group 21.3 (interquartile range [IQR] 14.8–46.7) years and 30.1 (IQR 10.1–41.8) for G+HDA group, but other demographic factors were similar. Overall, the cumulative incidence of CMV reactivation was significantly lower in the G+HDA group (60% vs. 96.7; p=0.001 [Gray's test]) (Figure 1). In patients receiving G+HDA, the median time to first positive CMV PCR occurred later (27 days [IQR 11–35]) when compared to those given SDA prophylaxis (17 days [IQR 8–25]) (p=0.26). Additionally, the G+HDA group had significantly lower initial (71 copies/mL [IQR 47–110] vs. 235 [IQR 63–760], p=0.006) and maximum PCR viral loads (VL) (170 copies/ml [IQR 88–310] vs. 3200 [1400-11000], p<0.001) when compared to those receiving SDA prophylaxis. In multivariate analyses, the G+HDA prophylactic strategy was also associated with a significant reduction in CMV reactivation (HR 0.31; 95% CI 0.16–0.58; p<0.001). Over the first 100 days following transplant, there were fewer episodes of invasive CMV disease in the G+HDA group (1/30, 3% [1 pneumonia]) than under SDA prophylaxis (5/31, 16% [1 disseminated, 2 pneumonia, and 2 GI]) (p=0.09). In the SDA group 2/5 (40%) patients died secondary to CMV disease, and an additional 2 patients developed fatal CMV pneumonia after day 100 (day 165 & 191); no CMV related death or cases of late disease developed in the group receiving G+HDA prophylaxis. There was no evidence of increased toxicity by either median and maximum creatinine levels or days to engraftment when comparing the two regimens. Conclusions: Our study demonstrates that G+HDA was effective in preventing CMV complications in UCBT recipients. This intensified prevention strategy was associated with a decreased rate of CMV reactivation and appeared to significantly alter CMV replication dynamics. Importantly, the increased valacyclovir exposure did not alter the risk for developing either renal or hematologic toxicity. Disclosures: No relevant conflicts of interest to declare.

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Comparison of Lengths of Stay for Inpatients with Immune Thrombocytopenia Treated with Anti-Rh(D), Intravenous Immunoglobulin, and Steroids

Blood ◽

10.1182/blood.v120.21.4646.4646 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4646-4646

Author(s):

Hrvoje Melinscak ◽

Ilan Shapira ◽

Mala Varma

Keyword(s):

Length Of Stay ◽

Hospital Stay ◽

Intravenous Immunoglobulin ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Average Length ◽

Length Of Hospital Stay ◽

P Value ◽

Onset Of Action ◽

Lengths Of Stay

Abstract Abstract 4646 Rationale: Anti-Rh(D) is an effective treatment for acute immune thrombocytopenia [ITP]. It has a faster onset of action (1 day) vis a vis corticoids (3 days) and intravenous immunoglobulin [IVIG] (4 days). A direct comparison of length of stay for adult inpatients receiving these therapies has hitherto not been performed. We hypothesized that the length of stay would be shortest for patients treated with anti-Rh(D). Methods: A retrospective chart review was conducted to assess length of stay in relation to treatments proffered for ITP. The defining diagnosis of ITP [coded 287.31] was rendered from the computerised record at St. Luke's-Roosevelt Hospital Center and identified 303 patients, of which 147 received treatments for active ITP within a period spanning 01SEP2005 through 29FEB2012. Treatments consisted of prednisone alone, dexamethasone alone, anti-Rh(D) alone, IVIG alone, and combinations of corticoids and the latter two. An average length of stay was tabulated for each treatment regimen. Age and gender were also recorded. Results: A total of 147 hospitalisations for ITP were noted and the analysis of variance statistical calculation applied thus. The median age was 48 years and the male:female ratio was 1.1:1. Eleven groups were delineated and the means for length of hospital stay with confidence intervals derived. The groups were as follows: Prednisone, Dexamethasone, Methylprednisolone, Anti-Rh(D), IVIG, Prednisone and Anti-Rh(D), Dexamethasone and Anti-Rh(D), Prednisone and IVIG, Dexamethasone and IVIG, Methylprednisolone and IVIG, and Anti-Rh(D) and IVIG or Dexamethasone, Anti-Rh(D), and IVIG. The overall p-value for length of stay was 0.0016 (Table 1). The shortest stays were recorded for the corticoid alone groups; however, the mean stay for anti-Rh(D) was shorter than that of IVIG, both in sole and combined modality treatments. Conclusion: Anti-Rh(D) is favourable with respect to hospital stay duration. Although corticoids result in still shorter lengths of stay, anti-Rh(D) demonstrated a shorter length of stay compared with IVIG. Combined with its single dosing and relative cost savings, anti-Rh(D) is an excellent alternative to IVIG. Disclosures: No relevant conflicts of interest to declare.

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Large Conserved Domains Of Low DNA Methylation Maintained By 5-Hydroxymethycytosine and Dnmt3a

Blood ◽

10.1182/blood.v122.21.2406.2406 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2406-2406

Author(s):

Mira Jeong ◽

Deqiang Sun ◽

Min Luo ◽

Yun Huang ◽

Myunggon Ko ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Hox Genes ◽

Conflicts Of Interest ◽

P Value ◽

Genomic Feature ◽

Conserved Domains ◽

Hematopoietic Stem ◽

Genome Wide ◽

The Impact

Abstract Identification of recurrent leukemia-associated mutations in genes encoding regulators of DNA methylation such as DNMT3A and TET2 have underscored the critical importance of DNA methylation in maintenance of normal physiology. To gain insight into how DNA methylation exerts the central role, we sought to determine the genome-wide pattern of DNA methylation in the normal precursors of leukemia cells: the hematopoietic stem cell (HSC), and investigate the factors that affect alterations in DNA methylation and gene expression. We performed whole genome bisulfite sequencing (WGBS) on purified murine HSCs achieving a total of 1,121M reads, resulting in a combined average of 40X coverage. Using Hidden Markov Model we identified 32,325 under-methylated regions (UMRs) with average proportion of methylation ≤ 10% and by inspecting the UMR size distribution, we discovered exceptionally large “methylation Canyons” which span highly conserved domains frequently containing transcription factors and are quite distinct from CpG islands and shores. Methylation Canyons are a distinct genomic feature that is stable, albeit with subtle differences, across cell-types and species. Canyon-associated genes showed a striking pattern of enrichment for genes involved in transcriptional regulation (318 genes, P=6.2 x 10-123), as well as genes containing a homeobox domain (111 genes, P=3.9 x 10-85). We compared Canyons with TF binding sites as identified from more than 150 ChIP-seq data sets across a variety of blood lineages (>10)19 and found that TF binding peaks for 10 HSC pluripotency TFs are significantly enriched in entirety of Canyons compared with their surrounding regions. Low DNA methylation is usually associated with active gene expression. However, half of Canyon genes associated with H3K27me3 showed low or no expression regardless of their H3K4me3 association while H3K4me3-only Canyon genes were highly expressed. Because DNMT3A is mutated in a high frequency of human leukemias24, we examined the impact of loss of Dnmt3a on Canyon size. Upon knockout of Dnmt3a, the edges of the Canyons are hotspots of differential methylation while regions inside of Canyon are relatively resistant. The methylation loss in Dnmt3a KO HSCs led Canyon edge erosion, Canyon size expansion and addition of 861 new Canyons for a total of 1787 Canyons. Canyons marked with H3K4me3 only were most likely to expand after Dnmt3a KO and the canyons marked only with H3K27me3 or with both marks were more likely to contract. This suggests Dnmt3a specifically is acting to restrain Canyon size where active histone marks (and active transcription) are already present. WGBS cannot distinguish between 5mC and 5hmC, so we determined the genome-wide distribution of 5hmC in WT and Dnmt3a KO HSCs using the cytosine-5-methylenesulphonate (CMS)-Seq method in which sodium bisulfate treatment convert 5hmC to CMS; CMS-containing DNA fragments are then immunoprecipitated using a CMS specific antiserum. Strikingly, 5hmC peaks were enriched specifically at the borders of Canyons. In particular, expanding Canyons, typically associated with highest H3K4me3 marking, were highly enriched at the edges for the 5hmC signal suggesting a model in which Tet proteins and Dnmt3a act concomitantly on Canyon borders opposing each other in alternately effacing and restoring methylation at the edges, particularly at sites of active chromatin marks. Using Oncomine data, we tested whether Canyon-associated genes were likely to be associated with hematologic malignancy development and found Canyon genes were highly enriched in seven signatures of genes over-expressed in Leukemia patients compared to normal bone marrow; in contrast, four sets of control genes were not similarly enriched. Further using TCGA data, we found that expressed canyon genes are significantly enriched for differentially expressed genes between patients with and without DNMT3A mutation (p value<0.05) Overall, 76 expressed canyon genes, including multiple HOX genes, are significantly changed in patients with DNMT3A mutation (p=0.0031). Methylation Canyons, the novel epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development. Disclosures: No relevant conflicts of interest to declare.

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Polymorphisms in the CYP450 Genes Influences Regimen Related Toxicity and Outcome in Patients with Beta Thalassaemia Major Undergoing HSCT.

Blood ◽

10.1182/blood.v114.22.869.869 ◽

2009 ◽

Vol 114 (22) ◽

pp. 869-869

Author(s):

Poonkuzhali Balasubramanian ◽

Salamun Desire ◽

Vikram Mathews ◽

Kavitha M Lakshmi ◽

Shaji R Velayudhan ◽

...

Keyword(s):

Risk Factors ◽

Annual Meeting ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

P Value ◽

Thalassaemia Major ◽

Hematopoietic Stem ◽

Beta Thalassaemia ◽

Variant Genotype ◽

The Impact

Abstract Abstract 869 Polymorphisms in drug metabolizing enzymes are known to contribute to inter-individual differences in the pharmacokinetics (PK) of the two most commonly used drugs for conditioning for hematopoietic stem cell transplantation (HSCT), busulfan (Bu) and cyclophosphamide (Cy) and their metabolites in plasma. We have previously reported the impact of CYP genes on the PK of Cy, [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 99] and the influence of Cy PK on transplant outcome [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 1820]. We have now extended this study to evaluate a total of 19 polymorphisms in 11 genes that are known to be involved in the metabolism of Bu and Cy. 180 of the 276 patients with thalassemia major who underwent HSCT between March 1991 and Dec 2008 and for who genomic DNA was available were included in the study. The following polymorphisms were screened using PCR followed by RFLP and/ or gel electrophoresis: GSTA1*B, GSTM1 and GSTT1 deletion, GSTP1*B, CYP2B6*2, *3, *4, *5 and *6, CYP2C9*2, *3 and *4, CYP2C19*2, *3, CYP3A4*1B, CYP3A5*3, *6 and ALDH1A1*2 and ALDH3A1*2. Polymorphism frequencies were associated with regimen related toxicities, other transplant related complications using Fischer's Exact test and Cox-proportional hazard's model.. Significant associations are shown in the Table. On univariate analysis, CYP2B6*4 variant genotype was associated with incidence of hemorrhagic cystitis (HC); CYP2C9*3 variant genotype was associated with the severity of HC; CYP2C19*3 and 2C9*2 genotypes were associated with overall and even-free survival (OS and EFS) and CYP2C9*2 and CYP2C9*3 genotype was associated with transplant related mortality (TRM). Multivariate analyses performed adjusting for known clinical risk factors still showed these genotypes to be significantly associated with outcome parameters. Variant genotypes of polymorphisms that result in decreased metabolism of Cy are protective against regimen related toxicities while these polymorphisms were risk factors for EFS and OS in the present study. This is the first report on the influence of common GST, CYP and ALDH polymorphisms on outcome of HSCT in patients with thalassaemia major. Screening for these polymorphisms in patients with beta thalassaemia undergoing HSCT can help identify patients at higher risk of complications.Table:EndpointGenotypeRelative risk (95% CI)P- value HCCYP2B6*4 variant0.3 (0.13-0.889)0.028 HC grade 1 vs. HC grade 2-4CYP2C9*3 variant0.2 (0.073-0.962)0.043 TRM2C9*2 variant2.7 (1.08-6.77)0.034 2C9*3 variant2.3 (1.0-5.7)0.049 OS and EFS2C19*3 variant3.3 (1.2-9.3)0.018 2C9*2 variant1.3 (0.93-2.03)0.070 Disclosures: No relevant conflicts of interest to declare.

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High-Dose Cyclophosphamide + G-CSF Mobilization Regimen Have Higher Anti-Myeloma Effects Than G-CSF Alone Regimen – Single Institutional Study in 147 Myeloma Patients From Japan

Blood ◽

10.1182/blood.v118.21.2007.2007 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2007-2007

Author(s):

Akira Tanimura ◽

Masataka Takeshita ◽

Atsushi Sato ◽

Junichiro Takano ◽

Hideaki Kitahara ◽

...

Keyword(s):

Disease Control ◽

Therapeutic Intervention ◽

Conflicts Of Interest ◽

Alkylating Agents ◽

Staging System ◽

Rank Test ◽

P Value ◽

High Dose ◽

Hematopoietic Stem ◽

Log Rank Test

Abstract Abstract 2007 Background: High-dose cyclophosphamide (HD-CY) + granulocyte-colony stimulating factor (G-CSF) and G-CSF alone have been used to mobilize hematopoietic stem cells (HSCs) for autologous SC transplantation (ASCT) in multiple myeloma (MM). However, which regimen is better is unknown; anti-myeloma effects of HD-CY + G-CSF have not been established. From January 1999 to June 2009, we administered HD-CY+G-CSF but changed to G-CSF alone during July 2009–December 2010. We retrospectively assessed HSC collection efficacy, complications, and anti-myeloma effects of these regimens. Patients and methods: We analyzed 147 MM patients from whom HSCs were to be collected at our institute. For mobilization, 115 patients were administered HD-CY (4 g/m2)+G-CSF (600 mg/body filgrastim or 500 mg/body lenograstim) and 32 were administered G-CSF alone (same dose as HD-CY). Here, 17 patients received therapeutic intervention between mobilization and transplantation without disease progression (PD). To avoid the patient outcome effect, we defined event- and progression-free survivals (EFS and PFS). EFS was defined as PD, death, or therapeutic intervention without PD. PFS was defined as PD or death, where therapeutic intervention without PD was used as a censor. Both were calculated from the start of mobilization. For analyzing response by mobilization, patients receiving therapeutic intervention without PD were excluded. Response was evaluated in those not receiving therapeutic intervention without PD or in whom response could not be evaluated before ASCT. Thalidomide was administered as maintenance therapy to 14 and 6 patients in the HD-CY+G-CSF and G-CSF groups after ASCT. Thalidomide administration was used as a censor. Results: Vincristine, doxorubicin, and dexamethasone (VAD) and HD dexamethasone (HDD) therapies were administered as induction therapy (VAD for 117, HDD for 2, and both for 11). New (bortezomib or thalidomide) and alkylating agents were administered to 7 and 13 patients, respectively. Before mobilization, 26 patients received radiotherapy; none were administered lenalidomide. No statistical difference was seen in baseline characteristics (Durie-Salmon stage, International staging system, interval from diagnosis to mobilization, disease control, and previous therapies) between both groups. However, patients mobilized by G-CSF alone were significantly older. Among 147 patients, 121 underwent planned ASCT. Of the 17 receiving therapeutic intervention without PD, 13 and 4 belonged to the HD-CY+G-CSF and G-CSF groups, respectively. More than 2 × 106 CD34-positive cells/kg were collected from 93% and 75% patients in the HD-CY+G-CSF and G-CSF (p = 0.0079) groups, respectively. More than 4 × 106 CD34-positive cells/kg were collected from 84% and 69% in the HD-CY+G-CSF and G-CSF (p = 0.07). Mean HSC count was 11.4 × 106/kg in the HD-CY+G-CSF group and 4.5 × 106/kg in the G-CSF group (p = 0.0007). Among patients receiving HD-CY+G-CSF, 66% were treated with intravenous antibiotics; 3 suffered cardiac shock and 2 septic shock. However, among those receiving G-CSF alone, no severe complications were seen. Median hospitalization days were 21 and 8 for the HD-CY+G-CSF and G-CSF groups, respectively (p < 0.0001). In the HD-CY+G-CSF group, 16% improved in disease control before ASCT, 71% showed no change, and 13% progressed. However, no patient improved, 63% showed no change, and 27% progressed in the G-CSF group (p = 0.015). Median EFS was 25 months in the HD-CY+G-CSF group and 13 in the G-CSF group (fig 1, p value of log-rank test = 0.012). Median PFS was 28 months in the HD-CY+G-CSF group and 15 in the G-CSF group (fig 2, p value of log-rank test = 0.011). Median overall survival did not differ significantly. Conclusion: Regarding the safety and duration of hospitalization, G-CSF alone may be safer and beneficial. However, HD-CY+G-CSF was more effective as a mobilization regimen and showed higher anti-myeloma effects than G-CSF alone. Disclosures: No relevant conflicts of interest to declare.

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Low Frequency of Molecular Alterations of H3.3-Atrx-Daxx Chromatin Remodeling Component Genes in Myelodysplastic Syndromes (MDS)

Blood ◽

10.1182/blood.v120.21.3844.3844 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3844-3844

Author(s):

Youmna Attieh ◽

Yue Wei ◽

Hui Yang ◽

Yu Jia ◽

Hong Zheng ◽

...

Keyword(s):

Dna Methylation ◽

Chromatin Remodeling ◽

Epigenetic Regulation ◽

Myelodysplastic Syndromes ◽

Conflicts Of Interest ◽

Cpg Island ◽

Genetic Mutations ◽

P Value ◽

Bone Marrow Mononuclear Cell ◽

Hematopoietic Stem

Abstract Abstract 3844 Novel sequencing technologies have allowed identification of a group of highly recurrent genetic mutations in myelodysplastic syndromes (MDS). Of importance, it has been noticed that a majority of these mutated genes in MDS encode important components of epigenetic regulation, including both DNA methylation and histone modifications. This phenomenon highlights the importance of epigenetic mechanisms in the pathogenesis of MDS. Recently, highly recurrent somatic mutations in the Histone H3.3-ATRX-DAXX chromatin remodeling pathway have been documented in pediatric glioblastoma (Schwartzentruber et al. Nature and Wu et al. Nature Genetics 2012), further supporting the importance of epigenetic regulation for tumorgenesis. We therefore examined potential genetic and epigenetic alterations of the same pathway in MDS. First, in a cohort containing 80 samples of MDS whole bone marrow mononuclear cell DNA (representative of both lower and higher risk disease), we performed Sanger sequencing covering genomic areas of reported mutations of H3F3A, H3F3B, ATRX, and DAXX in glioblastoma. Sequenced genomic areas included reported mutations in pediatric tumors: Lys27 and Gly34 of H3F3A and H3F3B; sequences upstream of and within the helices domain of ATRXX; and the whole coding sequence of DAXX. Overall, we only detected one mutation of H3F3A (K27N) in one MDS case (76 year old male with RA; INT-1; diploid). No other reported mutation of H3F3B, ATRX and DAXX genes was detected in any other patients of this MDS cohort. Because of the potential of epigenetic deregulation, we then examined status of DNA methylation for the promoters of ATRX and DAXX in MDS patients by bisulfite pyrosquencing. While no DNA hypermethylation of DAXX promoter was detected, 8 out of 40 (20%) patients had hypermethylation of the CpG island in the promoter region of ATRX. However, six of these eight patients were females. Based on reports of ATRX methylation in healthy females, it is likely that the 6 cases in female patients represent physiological × chromosome inactivation. Finally, we performed RT-PCR analysis using cDNA samples isolated from CD34+ hematopoietic stem cells of 40 MDS patients. Results indicated that expression of ATRX and DAXX were increased by 2 fold (p-value 0.07) and 5.2 fold (p-value 0.0003) respectively compared to control CD34+ cells. The implications of this phenomenon need to be studied further. Taken together, these results suggest that genetic mutations of the H3.3-ATRX-DAXX chromatin remodeling do not play a role in the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

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Ruxolitinib for Steroid-Refractory cGvHD: A Significant Response in Heavily Pretreated Patients after Haploidentical-SCT

Blood ◽

10.1182/blood-2020-141561 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-1

Author(s):

Hengwei Wu ◽

Jimin Shi ◽

Yi Luo ◽

Yamin Tan ◽

Mingming Zhang ◽

...

Keyword(s):

Safety Profile ◽

Conflicts Of Interest ◽

Janus Kinase ◽

P Value ◽

Severe Adverse Effect ◽

Fisher Exact Test ◽

Infection Prophylaxis ◽

Hematopoietic Stem ◽

Late Morbidity ◽

Steroid Refractory

Background Chronic graft-versus-host disease (cGVHD) is the leading cause of late morbidity and mortality as well as impaired quality of life after allogeneic hematopoietic stem cell transplantation (allo-SCT). Established first-line therapy for cGVHD still comprises corticosteroids and calcineurin inhibitors. However, about half of the patients with cGVHD are refractory to therapy with corticosteroids and the response rates ranges from 30% to 60%. No consensus has been reached regarding the optimal salvage treatment for steroid-refractory (SR)-cGVHD. Studies have reported that ruxolitinib (RUX, Jakafi, Novartis, Basel, Switzerland), a selective Janus kinase 1/2 (JAK1/JAK2) inhibitor, prevents GVHD but preserves the beneficial graft versus leukemia (GvL) effect. Here, we present study investigating the clinical efficacy of ruxolitinib and its safety profile in patients having SR-cGVHD after allo-SCT during the treatment course. Methods This was a retrospective study and all data were collected from the clinical history of a single center - the First Affiliated Hospital of Zhejiang University School of Medicine. Patients treated with ruxolitinib for SR-cGVHD after allo-SCT between August 2017 and December 2019 were involved. A two-tailed P value of 0.05 was considered statistically significant. Univariate comparisons of parameters were performed using the χ2 test, Fisher exact test, and Studentttest,as appropriate. A multivariate model was used to calculate the odds ratio (OR) with a 95% confidence interval (CI). Overall survival (OS) was estimated and plotted using the Kaplan-Meier method. Results A total of 41 patients with SR-cGVHD were eligible. The demographic and baseline characteristics of patients were summarized in Table 1, and GVHD information was presented in Table 2. After a median duration of 8 months (range, 1.1-24.9) of ruxolitinib treatment, the overall response rate (ORR) was 73.2% (30/41), including 15 patients (36.6%) with complete response (CR) and 15 patients (36.6%) with partial response (PR). Lung cGVHD (OR, 0.138; 95% CI, 0.024-0.803; P=0.028) and matched related donors (OR, 0.129; 95% CI, 0.020-0.835; P = 0.032) had independent adverse effects on treatment response. Major adverse events associated with ruxolitinib were cytopenias and infectious complications. Cytopenias were reported during ruxolitinib treatment in six patients (5/41, 12.2%), of which three were (7.3%) of grades 3-4. A total of 23 patients developed any grade infections: 19 Epstein-Barr virus (EBV) DNAemia, five cytomegalovirus (CMV) DNAemia, two carbapenem-resistant Klebsiella pneumoniae sepsis, and one hepatitis B (HBV) reaction. The 6-month and 12-month OS rates for all treated patients were 87.8% (95% CI, 77.3-98.3) and 61.0% (95% CI, 45.4-76.6), respectively. The median follow-up for this cohort was 14.9 months. Prolonged survival was observed in patients with a male donor, CR before transplantation, baseline moderate cGVHD, and skin cGVHD. Conclusion This cohort showed the efficacy of ruxolitinib in SR-cGVHD treatment with the ORR of 73.2% and CR rate of 36.6%. Despite the limited sample size and retrospective nature, the results of this study indicated that patients with no lung involvement and haploidentical relatives as donors were more likely to benefit from ruxolitinib. Regarding the safety profile, the present study showed that the infection event was the most severe adverse effect related to ruxolitinib, highlighting the significance of infection prophylaxis. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: ruxolitinib (RUX, Jakafi, Novartis, Basel, Switzerland)

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