bluuebunny/crossref_metadata_2025 huggingface dataset:
Relapse Rate and Long-Term Survival of AL Amyloidosis Patients Treated with High-Dose Melphalan and Autologous Stem Cell Transplantation (HDM/SCT).
Authors: Seldin David C. , Skinner Martha , Oran Betul , Quillen Karen , Finn Kathleen T. , Sanchorawala Vaishali
Published: October 2019

AL amyloidosis is a plasma cell dyscrasia in which clonal immunoglobulin light chains misfold and are deposited in tissues, leading to organ failure in untreated patients, with median survival of only ~1 year. Oral melphalan and prednisone is minimally effective for the disease, with an increase in median survival to ~1.5 years, and a low rate of hematologic complete responses (CRs). Twelve years ago, we began treating patients with AL amyloidosis with HDM/SCT. In the plasma cell malignancy multiple myeloma, this approach produces hematologic CRs and improves survival, but is not curative, as all patients eventually relapse. In AL amyloidosis, the relapse rate and long-term survival have not been studied, but early results are promising, with most centers reporting CR rates of ~40% and excellent survival in responding patients. To address the durability and long-term results of treatment with HDM/SCT, here we report on the outcome for AL amyloidosis patients treated at Boston Medical Center with HDM/SCT >10 years ago. The first autologous transplant took place on July 18, 1994 in two years, by July 18, 1996, 43 patients with AL amyloidosis without myeloma or other hematologic disease had been treated with HDM/SCT, receiving 100–200 mg/m2 melphalan depending upon age and protocol. Of the 43 patients treated in this 2 yr period, 76% of the patients were male, 81% had a lambda monoclonal disease, and their median age was 52 (range, 29–71). In these first 43 patients, the 100 day peri-transplant mortality rate was 16%. Nineteen of the 43 patients (44%) achieved a hematologic CR after treatment. In annual followup, 4 of 19 patients (21%) eventually relapsed. Fourteen of 43 patients (33%) are still alive; 12 of 19 patients who achieved a CR (63%) are still alive, while only 2 of 34 patients who did not (6%) are still alive. Although there were fewer (8) patients with kappa clonal disease, they had a better outcome, with an 87% CR rate vs. 34% for lambda (P=0.006); 75% of the kappa patients are still alive, vs. 23% of the lambda patients (P=0.005). The median survival of all 43 patients is 4.7 years. Thus, treatment of AL amyloidosis patients with HDM/SCT produces a high CR rate that is durable and is associated with excellent 10 year survival, particularly for those patients achieving a hematologic CR and for patients with kappa clonal disease. Ongoing clinical trials of HDM/SCT, along with strategies to reduce morbidity and mortality and to improve the CR rate, incorporating additional cycles of HDM/SCT or new anti-plasma cell agents, appear to be well-justified by these results.
High Proportions of CD4+CD25hi T Cells Pretransplant Correlate with Worse Overall Survival after Stem Cell Transplant.
Authors: Prieto-Hinojosa Adria , Madrigal J. Alejandro , Shaw Bronwen E. , Mayor Neema P. , Marsh Stephen G.E. , Travers Paul J. , Duarte Rafael F.
Published: October 2019

Regulatory cells may have a modulatory effect on alloreactive phenomena following hematopoietic stem cell transplantation (HSCT), although their role in this setting in patients remains controversial. We have analysed the effect of pre-conditioning peripheral blood levels of T-regs on the outcome of 89 adult patients undergoing HLA-identical (10/10) unrelated donor (UD)-HSCT. Allografts were T-cell depleted with Alemtuzumab, which has been described to spare T-regs from, and we found that higher proportion of CD4+CD25hi T-cells correlated with worse survival (p=0.002). This higher proportion also correlated with higher incidence of relapse (p=0.0274) and with higher incidence of chronic (c) GvHD (p=0.033) but not with the incidence of aGvHD.

Regulatory T cells (T-regs) are a naturally occurring regulatory population of CD4+CD25hi T-cells known to express the transcription repressor FoxP3 and to produce anti-inflammatory cytokines such as TGFβ and may therefore affect patient survival. We analyse the profile of the CD4+CD25hiT cells in our cohort. In our healthy donor control group (n=30), CD4+CD25hi-expression associated with both, FoxP3 mRNA-levels (r=0.649; p=0.001) and TGFβ production (r=0.912; p<0.001), as previously described for T-regs. In preconditioning patient samples however, CD4+CD25hi-expression had a strong correlation with TGFβ regulatory cytokine production (r=0.863; p<0.001) as expected. However, FoxP3 mRNA-expression correlated neither with the CD4+CD25hi T-regs phenotype (r=0.280; p=0.040), nor with the production of TGFβ (r=0.229; p=0.156), and appeared not be an accurate marker for regulatory T-cell function in this patient setting. In addition, an inverse correlation with TNFα expression (r=−0.458 p<0.001) was found that may argue that the CD4+CD25hi cells represent a true regulatory population and not activated cells. This suggests either that activated cells that are not expressing FOXP3 might be included in the CD4+CD25hi population or that CD4+CD25loFOXP3+ cells have not acquire the regulatory function (i.e. they are not expressing TGFβ).

In summary, CD4+CD25hi Regulatory T cells may have an impact suppressing allo-responses against the tumour, decreasing the overall survival by increasing the rates of relapse.

Although, in mice CD4+CD25hi population have been clearly identified by the expression of the FOXP3, which encodes a transcription repressor, in humans this remains controversial, where FOXP3 expression is not exclusive of the CD4+CD25hi population.

Relying on the expression of CD4+CD25hi and FOXP3 in patients is insufficient to determine regulatory T cell numbers and suggests that other parameters of regulatory function should be taken in to account.
Four Cases of Donor Cell-Derived Acute Myeloid Leukemia Following Unrelated Cord Blood Transplantation for Adult Patients; Tokyo Cord Blood Bank Experiences.
Authors: Nagamura-Inoue Tokiko , Kodo Hideki , Takahashi Tsuneo A. , Mugishima Hideo , Tojo Arinobu , Asano Shigetaka
Published: October 2019

Donor cell leukemia (DCL) is considered as a rare complication following allogeneic bone marrow transplantation (BMT), whereas the actual frequency of DCL has not yet been specified. Cord blood (CB) is now recognized as an alternative source for stem cell transplantation (SCT), with more than 6000 cord blood transplants (CBTs) performed worldwide, and a few cases of DCL following CBT have also been reported. Here we report four cases of DCL developed after unrelated CBTs using 490 shipped units from Tokyo cord blood bank (TCBB). Development of DCL was informed to TCBB by attending physicians of the recipients in CBT centers as soon as definite diagnosis was made. The feed-back from CBT centers on four DCL cases is summarized in Table 1. All the donors were well at the follow-up questionnaire 6–12 months after birth, but further information of their health conditions has not yet been obtained.

Table 1. Four cases of DCL following CBT Case 1 2 3 4 Recipient 32F 32F 56F 30M Disease AML in REL1 AML-M0 in REL1 ATL Hodgkin’s disease SCT 1st 2nd 2nd 2nd Regimen Myeloablative Myeloablative Non-myeloablative Non-myeloablative TBI 12Gy 12Gy (−) 2Gy G-CSF (+) (+) (+) (+) GVHD prophylaxis CsA+sMTX CsA+sMTX FK506+PSL CsA+sMTX aGVHD II II 0 III cGVHD (−) (−) (−) limited DCL AML AML-M2 AML AML-M5 Latent period 15 Mm after CBT 11 Mm after CBT 7 Mm after CBT 16 Mm after CBT Diagnosis FISH STR FISH STR

The etiology of DCL is unclear and a common mechanism is unlikely according to the reported literature. There exist several possibilities including occult leukemia or preleukemic state in the donor, defect in immune surveillance, therapy-related stromal abnormalities, excess of cytokine stimulation, and DNA replication and/or repair errors associated with post-transplant expansion of stem/progenitor cells. The possibility of occult leukemia in the donor raises serious problems regarding to the ethical responsibilities of the CBB to the donor.
Haploidentical Allogeneic Hematopoietic Cell Transplantation in Adults with Reduced-Intensity Conditioning and CD3/CD19-Depleted Grafts: Low Toxicity and Fast Engraftment.
Authors: Bethge Wolfgang A. , Faul Christoph , Bornhaeuser Martin , Stuhler Gernot , Lang Peter , Stelljes Matthias , Handgretinger Rupert , Kanz Lothar
Published: October 2019

Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with CD34-selected stem cells has been complicated by high regimen related toxicities, slow engraftment and delayed immune reconstitution leading to increased treatment related mortality (TRM). A new regimen using graft CD3/CD19 depletion with anti-CD3 and anti-CD19 coated microbeads on a CliniMACS device and reduced intensity conditioning may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, graft-facilitating- and dendritic-cells. Reduced intensity conditioning was performed with fludarabine (150–200 mg/m²), thiotepa (10 mg/kg), melphalan (120 mg/m²) and OKT-3 (5 mg/day, day −5 to +14) and no posttransplant immunosuppression. Twenty two consecutive patients (median age=43 (range, 21–59) years) have been transplanted with this regimen. Diagnosis were AML (n=12), ALL (n=5), NHL (n=2), MM (n=2) and CML (n=1). Patients were “high risk” with refractory disease or relapse after preceding HCT. The CD3/CD19 depleted haploidentical grafts contained a median of 7.6 x 106 (range, 3.4–17x106) CD34+cells/kg, 6.7x107 (range, 0.02–18.2 x107) CD56+cells/kg and 5.5x104(range, 0.006–44x104) CD3+T-cells/kg.

Donor-recipient KIR-ligand-mismatch was found in 14 of 22 patients. The regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis. Because of severe neurotoxicity in 4 patients treated with 200 mg/m² fludarabine, the dose was reduced to 150 mg/m² with no further cases of neuropathy thereafter. Engraftment was rapid with median time to >500 granulocytes/μL of 12 (range, 10–21) days, >20000 platelets/μL of 11 (range, 7–28) days and full donor chimerism after 2–4 weeks in all patients. Incidence of grade II–IV GVHD was 59% with grade II=9, III=2 and IV=2. One patient, who received the highest T cell dose, developed lethal grade IV GVHD. TRM in the first 100 days was 6/22 (27%) with deaths due to idiopathic pneumonia syndrome (n=1), mucormycosis (n=1), pneumonia (n=3) or GVHD (n=1). Six patients died after day 100, five due to relapse and one with systemic adenoviral infection. Overall survival is 10/22 patients (45%) with a median follow-up of 185 days (range, 17–988). In conclusion, this regimen is promising in high risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing.
No Impact of Adverse Karyotype on Outcome after Allogeneic Stem Cell Transplantation in Patients with High-Risk Acute Lymphoblastic Leukemia.
Authors: Tamm Ingo , Massenkeil Gero , Wagner Mandy , Terwey Theis , Lutz Christoph , Neuburger Stefan , Bommert Christiane , Dörken Bernd , Arnold Renate
Published: October 2019

Karyotypes like Philadelphia-chromosome (Ph+) and t(4;11) adversely influence clinical outcome in adult patients with acute lymphoblastic leukemia (ALL) after chemotherapy. Here, we analyze the potential impact of karyotype on outcome after allogeneic hematopoietic stem cell therapy (HSCT) in ALL.

We present a retrospective analysis of 135 adult ALL patients treated unicentrically within the German ALL (GMALL) protocol using allogeneic HSCT. All patients were high-risk patients in CR1 according to GMALL definitions (i.e., WBC > 30.000/μl, pro-B-ALL, t(4;11), t(9;22), early T- or mature T-ALL, no CR after first induction) or were beyond CR1.

Median age of all patients was 29 years (range 16 – 55), 99 B-lineage and 36 T-lineage ALLs were transplanted. Normal karyotype was present in 57 patients, Ph+ in 36 patients, complex chromosomal anomalies in six patients, t(4;11) in five patients, other aberrations in 22 patients and no growth/no cytogenetic data were available for 9 patients. Patients were transplanted in complete remission (CR) CR1 (60), CR2 (23), CR3 (7), first relapse (30), second relapse (6), third relapse (1) and eight patients had primary induction failure. 41 patients received bone marrow and 94 patients received peripheral blood stem cell transplants. Patients received standard high-dose (n=125) or reduced intensity conditioning (RIC) (n=10) HSCT from related (n=58) or unrelated (n=77) donors.

Overall, after a median follow-up of 11 months (range 1–120), 74 (55%) patients died and 61 (45%) are alive. Median follow-up of the living is 29 months (range 1–120). Deaths were due to treatment-related mortality (TRM; n=33; 24%) or relapse (n=41; 31%). Leukemia-free survival (LFS), overall survival, and TRM were not significantly different between the karyotype subgroups studied. LFS at 6 months, 1 year, 3 years, and five years was 62%, 60%, 50%, and 41% for patients with a normal karyotype; 63%, 47%, 40%, and 28% for patients with Ph+; and 61%, 40%, 27%, and 27% for patients with other aberrations. 4/5 patients with t(4;11) and 4/6 patients with complex chromosomal abnormalities are alive in CR.

In conclusion, there is no adverse impact of classical poor risk karyotypes on outcome within this high-risk group of ALL patients after allogeneic stem cell transplantation. In contrast, there was a trend that patients with t(4;11) and complex chromosomal anomalies did better (4/5 alive, 1/5 TRM and 4/6 alive, 1/6 TRM, 1/6 relapse, respectively) than patients with other aberrations (8/22 alive, 8/22 death due to relapse, 6/22 TRM). High-risk ALL patients with poor risk cytogenetics are candidates for allogeneic stem cell transplantation which can be curative. Whether other conditioning regimens or adoptive immunotherapy can reduce relapse rate in this patient group is a matter of ongoing clinical research.
Allogeneic Transplant Outcomes in Imatinib-Refractory Chronic Myeloid Leukaemia (CML) Are Similar to Transplant Outcomes in Imatinib-Responsive/Imatinib-Naïve CML and Appear To Be Predicted by the EBMT Risk Score.
Authors: Stylian Steven , Fennelly Eileen T. , Butler Jason P. , Morton James P. , Western Robyn , Hutchins Cheryl J. , Hill Geoff R. , Durrant Simon T. , Kennedy Glen A.
Published: October 2019

Aims: To review the outcome of allogeneic stem cell transplantation (SCT) in imatinib refractory chronic myeloid leukaemia (CML).

Methods: Outcomes of all allogeneic transplants performed after January 2001 for CML at our institution were retrospectively reviewed. Imatinib-refractory CML was defined as either lack of any cytogenetic response (CGR) after at least 6mths of imatinib, loss of CGR or progression to a more advanced disease stage (accelerated or blast phase) during imatinib therapy. Using the EBMT risk score (Lancet1998; 352: 1087), transplant outcomes for imatinib refractory CML were compared with all other CML transplants performed during the same time period. Survival analysis was performed using the Kaplan-Meier product-limit and comparison of survival data via the log-rank test.

Results: Of 31 allogeneic transplants (19M; 12F) performed for CML, 12 had been performed for imatinib refractory CML (no CGR to imatinib n=3; loss of CGR n=3; progression to AP n=3; progression to BC n=3), 5 in patients with imatinib responsive CML, and 14 in patients never exposed to imatinib. Median age at SCT was 40yrs (range 19–63yrs). Donor source included HLA-matched unrelated donors in 14 cases, HLA-identical siblings in 16 and other matched family donors in 1. Conditioning regimens included Cy/TBI (20 cases), Bu/Cy (8 cases), Flu/Mel (2 cases) and Flu/Cy (1 case); CsA + MTX was used as standard GVHD prophylaxis. EBMT risk scores were 1 (4 cases), 2 (6 cases), 3 (8 cases), 4 (5 cases), 5 (3 cases) and 6 (5 cases). At median follow-up post-SCT of 37mths (range 6–64mths), median PFS and OS are not reached; at 2yrs PFS, EFS and OS are 81%, 58% and 61% respectively. For patients with EBMT risk scores of 1–2 versus 3–4 versus 5–6, OS at 2 yrs post-SCT is 80%, 62% and 38% respectively (p=0.03). Based on EBMT risk score, no significant differences in PFS, EFS or OS were observed when comparing SCT for imatinib-refractory versus imatinib-responsive / imatinib-naïve CML.

Conclusion: Our experience suggests that survival post-SCT for imatinib-refractory CML is similar to SCT for imatinib-responsive / imatinib-naïve CML. The EBMT risk score appears to remain useful in predicting survival post-SCT in imatinib-refractory CML.
N-rasm-Transduced Fibroblasts Facilitate the Expansion and Engraftment of Umbilical Cord Blood Stem Cells.
Authors: Dolnikov Alla , Holmes Tiffany , Shen Sylvie , Knight Robert , O’Brien Tracey A.
Published: October 2019

Clinical trials using ex-vivo expanded umbilical cord blood (UCB) cells have thus far failed to demonstrate significant improvement in speed of hematopoietic recovery suggesting better understanding of self-renewal, proliferation, homing and engraftment capabilities of cord blood is needed. Recent progress has been made in the identification and understanding of the cellular niche for hematopoietic stem cells (HSCs) within bone marrow however attempts to identify soluble factors that regulate HSC engraftment to bone marrow have been less successful. We have demonstrated that N-rasm transduced NIH 3T3 fibroblasts release soluble factors that support the ex-vivo expansion of UCB CD34+ hematopoietic progenitor cells (HPCs). UCB CD34+ cells were cultured in combination with cytokines : TPO, SCF, IL-6 and flt3-ligand(all at 100ng/ml). Significant (7-fold) expansion of total nucleated cells was seen on day 19 in cultures complemented with medium produced by N-rasm -transduced fibroblasts (N-rasm medium), compared to control cells (GFP- transduced fibroblasts (GFP medium)). Maximum expansion of UCB CD34+ cells (2-fold) and the most primitive sub-population of CD34+ CD117+ cells (2-fold) were seen on day 4 in N-rasm medium cells compared to GFP medium cells. Furthermore, increased numbers of cycling cells were observed in UCB CD34+ cell cultures supplemented with N-rasm medium. Significant (3-fold) expansion of CD34+CXCR4+ cells enriched with the NOD/SCID mice engrafting cells, was also observed. This increased expansion of CD34+CXCR4+ cells correlated with a 3-fold increased engraftment of human UCB cells in a NOD/SCID mouse model confirming retention of engraftment capability of expanded cells. This data demonstrates that N-rasm -transduced fibroblasts secrete soluble factors that promote ex-vivo expansion and in-vivo engraftment of primitive human hematopoietic progenitor/stem cells derived from UCB. Proteins that are secreted by N-rasm -transduced fibroblasts that activate hematopoiesis are currently being identified. Our data demonstrates the potential of N-rasm-transduced fibroblasts as a reservoir for the discovery of novel secreted proteins that regulate ex-vivo expansion and engraftment of human UCB HSCs and may represent a potential novel expansion strategy allowing greater use UCB as a stem cell source for allogeneic transplantation.
Comparison of Donor Cell Homing Efficiency between Intraosseus and Intravenous Injection in Primate Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Qi Feng , Phua Cindy M.L. , Tan Peik K. , Chow Pierce K.H. , Hwang William Y.K.
Published: October 2019

The traditional route for the delivery of hematopoietic stem cells (HSC) for transplantation by intravenous (IV) injection results in only a small percentage of the injected cells homing to the bone marrow (BM) microenvironment - where hematopoiesis could be more optimal. As such, delivery of HSCs by intra-BM injection has been investigated using syngeneic mice or autologous primate transplants. However, there has been no animal study of allogeneic HSCT directly comparing intra-BM and IV injection in terms of the seeding of donor cells in recipient BM, peripheral blood and other organs. We present the first direct comparison of donor cell homing efficiency between intra-BM and IV injection in an non-human primate (Cynomolgus Monkey) model of allogeneic hematopoietic stem cell transplant (HSCT) model using a preparative regimen modified from existing clinical protocols. Two recipients with similar age, weight and height received pre-transplant conditioning chemotherapy comprising 30mg/m2 fludarabine from Day-5 to Day-3, and 50mg/kg cyclophosphamide from Day-5 to -2. Fresh BM cells from an unrelated donor were harvested and labeled with PKH26 cell membrane dye (labeling efficiency: 99.6%) on day 0. With the recipients under general anesthesia, 2×108 donor cells were transplanted into the conditioned recipients by IV or intra-BM injection using a modified syringe suction method. Intra-BM injection recipients were found to have significantly more donor PKH26 cells, compared with IV injection recipients, in recipient BM at 2 hours (6.9% versus 2.5%, p<0.01) and 4 hours (6.7% versus 2.2% p<0.01) after transplantation. There were somewhat fewer donor cells in the peripheral blood after intra-BM delivery (3.4% versus 4.6%) at 2 hours, though the percentages were the same 4 hours after injection (1.6% in both). Interestingly, donor cell percentages in the bone marrow remained stable between 2 to 4 hours after transplant, as opposed to donor cell percentages in the peripheral blood, which fell by 2.1 to 2.9 times between 2 to 4 hours after transplant. Furthermore, compared to IV injection, intra BM injection retained more cells within BM, with less cells trapped in other organs, like the spleen (1.4% vs. 3.1%) and liver (0.8% vs. 3.5%), as well as non-hematopoietic organs like the heart (1.1% vs. 4.9%), lung (1.0% vs. 4.0%) and kidney (1.5% vs. 3.2%). Our study showed that in a allogeneic non-human primate model of HSCT, intra-BM injection of HSC resulted in superior retention of donor cells in BM with reduced seeding onto non-hematopoietic organs - suggesting possible applications for improved donor cell engraftment and reduced effects of donor-cell-mediated graft versus host disease on non-hematopoietic organs.

Figure Figure
Dendritic Cell Adenosylhomocysteine Hydrolase-Like Protein 1 (AHCYL1) and Inositol Phospholipid Signaling Are Potential Targets for Modulating Their Function.
Authors: Kato Masato , Key Brian , Carter Adrian , Angel Nicola Z. , Cooper Benjamine J. , Hart Derek
Published: October 2019

Recipient and donor dendritic cells (DC) initiate acute graft versus host disease after allogeneic hematopoietic stem cell transplantation and activated DC predict for severe disease. Using a differential display technique, we isolated the cDNA for an adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), a novel intracellular protein with ~50% protein identity to adenosylhomocysteine hydrolase (AHCY) and showed that it was upregulated in activated human DC (Dekker et al. Immunogenetics.2002;53:993). AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor (IP3R), suggesting that AHCYL1 is involved in intracellular calcium release (Ando et al. J Biol Chem.2003; 278:10602). Given that intracellular calcium levels control DC function, we reasoned that AHCYL1 was a potential target for modulating DC function. Therefore, we sought functional data using the zebrafish model. We identified two zebrafish AHCYL1 orthologs (zAHCYL1A and B) by bioinformatics and reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative RT-PCR showed that zAHCYL1A and B mRNA expression was regulated differently to the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligos against zAHCYL1A and B into zebrafish embryos inhibited zAHCYL1A and B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies, that were similar to those obtained by depleting intracellular calcium with thapsigargin. The injection of hAHCY had little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in zebrafish embryogenesis by modulating IP3R function and consequent intracellular calcium release. It also suggests that blocking AHCYL1 in DC may have a significant effect on DC function, which might, ultimately, be exploited therapeutically. We have generated AHCYL1 gene deleted mice and these will be used to explore these questions further.
Pre-B Cell Acute Lymphoblastic Leukemia Cells Carrying a Bcr/Abl Translocation Retain Sensitivity to Minor Histocompatibility Antigen Specific T Cells but Are Resistant to Natural Killer Cells: Implications for Manipulation of Graft Versus Leukemia Effects.
Authors: Mullen Craig A. , Campbell Andrew , Young Faith , Jordan Craig T.
Published: October 2019

Background: Some acute lymphoblastic leukemias with high risk genetic lesions such as bcr/abl are treated with allogeneic BMT. Results of allogeneic BMT generally have shown only a modest improvement over high-dose chemotherapy. The graft versus leukemia (GVL) effect associated with allogeneic BMT is generally considered to be less potent in ALL than in myeloid leukemias. One hypothesis explaining reduced sensitivity of lymphoblastic leukemia cells to GVL is that such cells may be intrinsically resistant to the effects of cytolytic T or NK cells in the posttransplant immune environment.

Methods: Primary pre-B ALL leukemias with high risk human genetic lesions were induced by incubating bone marrow from mice null at the Ink4A/Arf locus with an ecotrophic retroviral vector containing a human bcr/abl fusion oncogene as well as a GFP marker. GFP+ leukemia cells were isolated and tested for sensitivity to T cells and NK cells. Minor histocompatibility antigen (mHA) specific T cells were generated by immunizing C3H.SW mice with irradiated C57BL/6 cells. NK cells were magnetically isolated from normal spleen cells using an anti-DX5 antibody. Leukemia cell populations were measured using a flow cytometry assay. 104 GFP-expressing leukemia cells were incubated with nonfluorescent T or NK cells in 96 well plates. At 48h leukemia cell population size was measured using fluorescent microspheres for quantitative internal controls. The percent survival of leukemia cells was expressed as a fraction of the absolute number of leukemia cells recovered from wells in which no cytolytic cells were present. In vivo sensitivity of the leukemia cells was measured by performing allogeneic BMT using donors that were naive or that had been previously sensitized to mHAs. 104 leukemia cells were infused at the time of transplant and survival and leukemia cell burden.

Results/Conclusions: Pre-B ALL cells exhibited exquisite sensitivity to the cytotoxic effects of anti-mHA T cells. In contrast, the lymphoblastic leukemia cells were resistant to the effects of NK cells. In vivo survival was prolonged and leukemia burden was reduced in those animals transplanted with specific anti-mHA T cells. These results suggest that mHA genes expressed in lymphoblastic leukemia cells may be suitable targets for post-transplant immunotherapy.
Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.
Authors: Scaramuzza Samantha , Trifari Sara , Marangoni Francesco , Martino Silvana , Metin Ayse , Galy Anne , Naldini Luigi , Aiuti Alessandro , Villa Anna , Dupré Loic , Roncarolo Maria Grazia
Published: October 2019

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.
Analysis of Health-Related Quality of Life in Patients Receiving AMG 531 Using the Disease-Specific Immune Thrombocytopenic Purpura Patient Assessment Questionnaire.
Authors: George James N. , Bussel James B. , McMillan Robert , Guo Matthew , Okano Gary J. , Nichol Janet L.
Published: October 2019

AMG 531 is a novel thrombopoeisis-stimulating peptibody that increases platelet production by targeting the thrombopoietin receptor. The trial described here is an ongoing, open-label, extension study of patients with immune thrombocytopenic purpura (ITP) assessing the safety and efficacy of long-term AMG 531 treatment of ITP, as well as patient-reported health-related quality of life (HRQOL). Eligible patients have completed a previous AMG 531 study in ITP. Patients previously treated with AMG 531 receive a starting dose that is the same as the final dose given in the previous study; placebo-treated patients begin the extension study with an AMG 531 dose of 1 μg/kg. The dose is skipped, decreased, maintained, or increased based on platelet count. Analysis was performed on the 36 patients (25 female, 11 male; 29 white, 6 Hispanic, 1 black) for whom complete data are currently available. The mean age is 50.0 ± 13.0 (SD) years. Most patients (83%) had a splenectomy prior to this study. HRQOL is being assessed with an ITP disease-specific measure, the ITP Patient Assessment Questionnaire (ITP-PAQ). This report describes the first use of the ITP-PAQ to measure treatment effect. The ITP-PAQ has 10 scales: 4 Physical Health (PH)—Symptoms, Fatigue, Bother, and Activity; 2 Emotional Health (EH)—Psychological and Fear; 3 Quality of Life (QOL)—Overall, Social, and Work; and 1 Women’s Reproductive Health (WRH). Each scale is scored 0 (worst)—100 (best). A planned interim analysis was conducted to evaluate changes in HRQOL from week 1 (pretreatment) to week 24. Results indicated improvement in the areas of PH-Symptoms, with a 7.9-unit increase (p=0.007), PH-Fatigue, with a 7.8-unit increase (p=0.006), PH-Bother, with a 9.4-unit increase (p=0.025), PH-Activity, with a 7.4-unit increase (p=0.032), and QOL-Work, with a 7.9-unit increase (p=0.014). An analysis of minimal clinically important difference will be conducted when the sample size is large enough to determine if statistically significant values are clinically meaningful. A comparison of HRQOL in patients with a durable platelet response (doubling of the baseline count and ≥50×109/L at 6 or more weeks during weeks 17–24 in the absence of rescue medication) versus those without a durable response showed a trend for greater improvement among the responders in all 10 scales, with significant differences for PH-Symptoms (p=0.02), PH-Bother (p=0.02), PH-Activity (p=0.01), EH-Psychological (p=0.01), and QOL-Overall (p=0.03). Individualized dosing of AMG 531 provides a unique therapeutic option for managing platelet count in ITP while improving HRQOL.
Advantages wth Using the Levonorgestrel-Releasing Intrauterine System in Females with Hemostatic Disorders.
Authors: Lukes Andrea S. , Arepally Gowthami
Published: October 2019

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is a small device placed within the uterus that is used for both contraception and treatment of menorraghia. This system releases 20 micrograms of levonorgestrel every 24 hours over 5 years. We hypothesized that females with inherited or acquired bleeding disorders would have decreased menstrual bleeding with the LNG-IUS and improved quality of life.

METHODS: A small, retrospective case series of females with hemostatic disorders who used the LNG-IUS within a 2 year period and who were seen at a tertiary medical center.

RESULTS: Seven females were treated with the LNG-IUS for menorrhagia and contraception. We did not reverse anticoagulation for placement of the device and there were no bleeding complications associated with the LNG-IUS insertion. The average age was 36.7 years (range 28 – 48 years). Four females had von Willebrand’s disease (VWD). Four females were on antithrombotic therapy, 2 warfarin and 2 aspirin. One female with VWD was heterozygous for factor V Leiden and was on warfarin for treatment of deep venous thrombosis. Six females had menorrhagia and one had bleeding most of the time. The average days of menstrual bleeding prior to LNG-IUS was 8.3 days which improved to 4.5 days after placement of the LNG-IUS. Three of 7 females discontinued use of the LNG-IUS, yet one had the LNG-IUS removed because she no longer required contraception. This female became pregnant and after an uncomplicated normal spontaneous vaginal delivery elected to use the LNG-IUS again for contraception and the benefit of decreased menstrual bleeding. The other 2 females (2/7, 29%) discontinued use of the LNG-IUS because there was worsening of heavy menses or ‘failed’ therapy - one with VWD and one on anticoagulation. The female on anticoagulation in this case also had significant uterine fibroids. A ten item quality of life questionnaire developed by Kouides was administered, and showed overall improvement in quality of life.

CONCLUSION: The levonorgestrel-releasing intrauterine system is an effective method of contraception and an effective treatment for heavy menses. For females with hemostatic disorders, this device has unique advantages: it is technically easy to use in females at increased bleeding risk, offers effective control of menorrhagia, and may likely improve overall quality of life. Additional prospective large scale studies are required to validate these findings.
Follow-Up of Patients with Curable Lymphomas Ouside of Clinical Trials: A National Survey of Practice Patterns.
Authors: Imrie Kevin R. , Rohlehr Yvonne , Cheung Matthew C.
Published: October 2019

With increasingly effective therapy for patients with aggressive histology non-Hodgkin’s Lymphoma and Hodgkin’s Lymphoma, more patients are attaining complete remission and are eligible for follow-up monitoring. Such monitoring offers the potential for early detection of relapse and surveillance for late effects of treatment, but is resource-intensive and may lead to unnecessary investigation of false relapses and cumulative radiation exposure. A number of Practice Guidelines (PG) provide recommendations on follow-up of patients with lymphoma, but provide conflicting recommendations particularly relating to the use of imaging. The PG of the American College of Radiology (ACR) and the National Comprehensive Cancer Network (NCCN) recommend routine use of imaging in follow-up, while those of the Canadian Association of Radiologists (CAR) and Cancer Care Ontario (CCO) recommend limiting imaging to patients suspected of relapse and those at higher risk of recurrence. We conducted a survey of practicing hematologists across Canada to document follow-up practice of patients outside of clinical trials. A 12-question survey was mailed to 244 Hematologists identified from the database of the Royal College of Physicians and Surgeons of Canada. The Dillman method was used to administer the questionnaire with a second mailing sent at 4 weeks to non-respondents. The target response rate was 50%. 127 responded (52%) after the second mailing. 93% indicated that they routinely follow-up patients in remission following curative treatment. 41% reported following patients indefinitely, 39% followed patients for up to 5 years, and 22% tailored the duration to the risk of relapse (generally 2–5 years). The median visit interval in years 1–2 was 3 months, years 3–5 6 months, and beyond 5 years 12 months. 41% used imaging (generally CT scans) routinely in all patients, while 31% used scans only in patients in partial remission and 21% in patients felt to be high risk at presentation. The majority of respondents performed CBC and LDH blood testing routinely in all patients and performed serum TSH levels in patients following mediastinal radiation. Serum electrolytes, liver function testing and blood films were ordered routinely only by a minority of respondents. 27% of practitioners reported utilizing printed educational material for patients and/or primary care practitioners regarding follow-up. 25% indicated an intent to change their follow-up practice in the near future, generally citing a plan to limit the duration of follow-up and to decrease reliance on CT scanning.

Conclusion: This national survey indicates that the majority of Canadian hematologists routinely follow patients with lymphoma after curative treatment. The use of imaging in follow-up is variable, but is more consistent with the recommendations of the CAR and CCO than the ACR and NCCN.
Long-Term Follow-Up of Monoclonal Gammopathies in Gaucher Disease Patients on Enzyme Replacement Therapy.
Authors: Park Jae H. , Moore Allan F. , Kuter David J.
Published: October 2019

Gaucher Disease is a lysosomal storage disease in which glucocerebroside accumulates in macrophages because of a deficiency of the lysosomal enzyme, β-glucocerebrosidase. Polyclonal and monoclonal gammopathies (MG) have been frequently associated with long-standing Gaucher Disease, and there have been reports suggesting an increased incidence of multiple myeloma (MM) in these patients. Chronic stimulation of the immune system secondary to progressive glucocerebroside storage and increased expression of cytokines such as Interleukin 6 have all been postulated to be the mechanism underlying this association. The purpose of this study was to ascertain the incidence and natural history of MG in Type 1 Gaucher Disease patients on enzyme replacement therapy (ERT) followed for up to 15 years. Twenty-eight patients with Type I Gaucher Disease on ERT seen at a single medical center were selected for the analysis. Of the twenty-eight patients, 17 (60.7%) were male; 11 (39.3%) were female; and the median age at the initiation of therapy was 42 years (range 25–85 years). Serum protein electrophresis (SPEP), immunofixation, and immunoglobulin quantitation were performed in all of these patients before initiating ERT and at intervals during follow-up. The patients were categorized into three groups based on their initial SPEP results: Group 1: those with normal SPEP pattern; Group 2: those with polyclonal gammopathy; and Group 3: those with MG. At the time of initiation of ERT, Group 1 contained 12 patients (42.8%) while Groups 2 and 3 each had 8 patients (28.6%). During follow up for a median of 8.5 years (range 1–15 years), 2 patients from the Group 1 (16.7%) and 4 patients from Group 2 (50%) developed MG. Of the patients in Group 3, 3 patients (21.4%) reverted to a normal SPEP pattern. Overall, 14 patients (50%) in all three groups had a MG identified at the time of initiation of therapy or during follow-up: 3 (all in Group 3) reverted to a normal SPEP pattern while the other 11 patients showed no change in M-spike levels after a median of 8.5 years of follow up. All 14 patients with MG had a monoclonal IgG immunoglobulin, and 3 of them also had a monoclonal IgM immunoglobulin. There was no association between the occurance or type of MG with any particular Gaucher Disease genotype, patient age, or other clinical variable. Only one of the patients developed a malignancy: an elderly man with MG developed Hodgkin’s Disease. None of our patients developed MM. In conclusion, these data confirm the high rate of MG in patients with Gaucher Disease but a low rate of progression to any other plasmacytic or lymphocytic disorder.
The Use of Erythropoietic Agents in Patients with Non-Myeloid Hematological Malignancies.
Authors: Shehata Nadine , Walker Irwin , Meyer Ralph , Haynes Adam , Imrie Kevin
Published: October 2019

Practice guidelines for use of an erythropoietic agent (EpA) have been previously developed for patients with all cancers, but these have not specifically addressed non-myeloid hematological malignancies. Given issues of benefit, cost, access to treatment and practice variation for these patients, the Cancer Care Ontario Program in Evidence - Based Care (CCO PEBC) conducted a systematic review and has developed a practice guideline. Entries to MEDLINE, CANCERLIT, EMBASE, the Cochrane Library databases, and abstracts of the American Societies of Clinical Oncology (ASCO) and Hematology (ASH) were searched. A hierarchy of outcomes was developed that included survival, quality of life (QoL), transfusion requirements and improvements in hemoglobin concentration/hematocrit. To validate conclusions and recommendations, the practice guideline was sent to Ontario practitioners in July 2006; responses are now being collated. Eighteen reports (14 articles, 4 abstracts) of randomized trials met eligibility criteria. None of the 3 trials reporting survival outcomes detected a benefit with use of an EpA. Of 7 trials evaluating QoL, 6 reported superior outcomes in patients receiving an EpA. None of those trials sufficiently reported methodologic parameters required by proposed guidelines for analyzing, interpreting and reporting QoL measures. Use of an EpA significantly reduced the proportion of patients transfused in 5 trials evaluating this outcome; reductions ranged from 15% to 40%. The absolute risk reduction ranged from 15% to 24%; the number needed to treat to prevent a transfusion ranged from 4 to 6. Use of an EpA was not associated with a reduction in the mean/median number of units transfused. In 10 studies, either a statistically significant increase in the hemoglobin concentration/hematocrit or in the proportion of patients with an increase in the hemoglobin concentration/hematocrit was seen. For this practice guideline, we interpreted the evidence as providing insufficient data to justify use of an EpA in order to improve survival or QoL. However, there are compelling data showing that use of an EpA reduces the risk of requiring a transfusion. Initiatives such as the Commission of Inquiry on the Blood System in Canada (“the Krever Commission”), state that alternatives to transfusion be offered to patients because of associated known and potentially unknown adverse consequences of blood products. Thus, the use of an EpA is recommended as an alternative to transfusion. However, the decision to use an EpA to reduce transfusion requirements should primarily consider individual patient values and the likelihood that a patient will require a transfusion so that patients are not exposed to unnecessary treatment and the health care system to additional costs.
Treatment of Osteopenia/Osteoporosis after Allogeneic Hematological Cell Transplantation with Zoledronic Acid.
Authors: Hill Wolfgang , Ullmann Johanna , Baur Andrea , Ledderose Georg , Tischer Johanna , Muehlbayer Dieter , Hausmann Andreas , Schmidt Michael , Haus Ulrike , Kolb Hans-Jochem
Published: October 2019

Decreased bone mineral density (BMD) has been found to be a relatively common complication following haematopoietic cell transplantation (HCT). Only some studies exist about therapy with antiresorptive substances such as bisphosphonates. These studies analyse either short time therapy or only the use for female patients (pts). The aim of this study was to test the effect and tolerance of zoledronic acid in long time survival pts after HCT. Methods: 40 pts with osteoporosis (WHO T-score<=−2.5 SD) or osteopenia (T-score −1,0 to −2.5) were included in this prospective phase II study. The dose was 4 mg intravenously all 3 months for 2 years (yrs) in combination with calcium, vitamin D and hormone-replacement therapy. 36 patients were evaluated, who got at least 3 doses (24 male, 12 female; mean age 43,8 yrs). The diagnoses were AML (15pts), ALL (5pts), CML (11pts), NHL (3pts), MDS (2pts). 16 pts received peripheral stem cells, 16 bone marrow, 4 pts both (median 15,6 months since HCT). All pts were evaluated according biochemical routine parameters, deoxypyridinoline (Dpd) and calcium excretion in urine by longitudinal measurements. The BMD of lumbar spine L1–L4 (LS) and total femur hip (FH) was mostly determined by dual-energy X-ray absorptiometry (Lunar Prodigy). 26 pts had at least two measurements since base line beginning of the treatment. Results: We found a significant increase of BMD at LS and FH (Table 1). The increase was better in younger pts <45yrs than in older pts at FH: 8,9 vs 2,9 % at 1 yr and 12,0 vs 3,4% at 2 yrs (p=0,007 and p=0.02). Dpd decreased from 6,2 nmol/mmol creatinine to 3,6 after 1 yr (p=0.009) and 3,2 after 2 yrs (p=0.04). By subgroup evaluation no significant changes of BMD were observed by testing pts with (n=20) or without (n=6) corticosteroid therapy for GvHD before or during the study (n= 7 and n=19) and by testing pts with osteoporosis (n=13) vs osteopenia (n=13), sex differences, diagnoses or time to HCT. Zoledronic acid was well tolerated. 1 of the 40 pts was excluded after the fist dosis because of dramatically myalgia. 6 pts had mild flu-like syndromes, bone pain, chest pain or headache. Zoledronic acid increases BMD and reduces Dpd excretion significantly in patients with bone loss after allogeneic hematological cell transplantation.

Table 1: baseline ° 1 yr°* 2 yrs°* % 1 yr* % 2 yr* °g/cm² mean values (+/−SD) *p=0.000 vs baseline LS 0.97+/−0.15 1.04+/−0.18 1.10+/−0.18 7.4+/−4,8 11.6+/−6.0 FH 0.82+/−0.10 0.87+/−0.11 0.91+/−0.10 5.6+/−5.3 7.5+/−7.0