bluuebunny/crossref_metadata_2025 huggingface dataset:
Appearance of Stress-Inducible Membrane Proteins ULBP on Blood Cells of Patients with PNH, Aplastic Anemia, and MDS: Possible Implication of ULBP in Immune-Mediated Marrow Injury.
Authors: Hanaoka Nobuyoshi , Horikawa Kentaro , Tsuzuki Yasuchika , Nagakura Shoichi , Ishihara Sonoko , Mitsuya Hiroaki , Nakakuma Hideki , Kawaguchi Tatsuya
Published: April 2020

Although it has been suggested that paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA), and myelodysplastic syndromes (MDS) share immune-mediated bone marrow injury, the molecular events in the injury is still unclear. Currently we have shown that leukemic K562 cells became less sensitive to natural killer (NK) cells in vitro when the leukemic cells acquired PIG-A mutations (Nagakura S et al, Blood2002; 100: 1031–1037). The decreased sensitivity was explained by a deficiency of stress-inducible and GPI-linked membrane proteins ULBP (Hanaoka N et al, Blood2004; 104: 774a). In general, the exposure of ULBP on cell membrane leads to activation of both NK and T cells expressing NKG2D, a receptor for ULBP. It is then conceivable that the ULBP-NKG2D engagement causes blood cell injury. In support of this idea, we detected both ULBP-expressing (ULBP+) blood cells (granulocytes or marrow cells) in 9 of 17 (53%) patients with PNH and killing of granulocytes of 4 the 9 patients by autologous lymphocytes. Granulocytes of healthy donors were negative for ULBP and intact in the killing assay. ULBP+ granulocytes were further detected in 24 of 45 (53%) patients with AA, and 10 of 21 (48%) patients with MDS, whereas ULBP+ granulocytes were detected in neither patients with autoimmune hemolytic anemia nor those with drug-induced marrow injury. Our study suggests not only that there was pathogenic stress to induce ULBP on blood cell membrane but also that the ULBP-NKG2D engagement was implicated in the pathogenesis of marrow failure, at least in a part, of patients with PNH and its related disorders. We thus propose that membrane ULBP would be of potential probe for the diagnosis of immune-mediated marrow injury and the evaluation of its treatment.
Risk Factors for Survival and Thrombosis in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH): A Single Institution Study of 68 Patients.
Authors: Smith Sean B. , Tefferi Ayalew , Hoyer James D. , Wolanskyj Alexandra P.
Published: April 2020

Background:

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal stem cell disorder characterized by intravascular hemolysis, thrombosis, bone marrow failure and reduced survival. In this current single institution study of patients with PNH, we investigate the risk factors predictive of survival and thrombosis.

Methods:

Data were abstracted from medical records of a consecutive cohort of patients with PNH seen at the Mayo Clinic. Diagnosis was confirmed by the presence of intravascular Hemolysis and either a positive Ham’s test, and/or by the presence of a GPI-deficient hematopoietic clone. Follow-up data were obtained by contacting the patients and by reviewing the social security death index.

Results:

i. Patients and treatment:

The study cohort included 68 patients with PNH (median age 32 years, range 10–77; 33 females) with a median follow up of 84.5 months (range, 1–538 months). Presenting features included infection (n=9), thrombosis (n=9), neutropenia (absolute neutrophil count (ANC) < 1000) (n=32) severe intravascular hemolysis (n=16) and splenomegaly (n=11). Prior to the diagnosis of PNH, 6 patients (8.8%) carried a diagnosis of aplastic anemia (AA) and 2 (2.9%) myelodysplastic syndrome (MDS). Complications documented after diagnosis included major thrombosis (n=11), major hemorrhage (n=4), and development of AA (n=7), MDS (n=4), and non-hematologic cancer (n=4) but none experienced leukemic transformation. Treatment included prednisone (n=30), other immunosuppressive agents (n=6), androgens (n=10), and bone marrow transplantation (n=4). Review of treatment with systemic anticoagulation disclosed only 2 patients that were receiving prophylactic warfarin therapy and both patients remain free of thrombosis after 1 and 2 years of therapy.

ii. Thrombotic events and risk factors:

Overall, 20 patients (29.4 %) experienced one or more episodes of major thrombosis. Among several clinical and laboratory parameters evaluated at presentation, only the detection of urine hemoglobin and infection were found to be significantly associated with the occurrence of thrombosis (p=0.05)

iii. Risk factors for survival

To date, 18 patients (27%) have died and median survival for the study cohort is projected at 21.1 years. Multivariate analysis identified thrombosis (p=0.003) and an ANC of > 1000 (p= 0.05) at presentation as independent risk factors for inferior survival. In addition, the occurrence of major hemorrhage was significantly associated with poor survival (p= 0.04).

Conclusion

The current study provides natural history information in a relatively large cohort of patients with PNH and identifies certain disease features that could be further explored for their potential as risk factors for both thrombosis and survival.
Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.
Authors: Smith Melody M. , Giver Cynthia R. , Waller Edmund K. , Flowers Christopher R.
Published: April 2020

Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect.

Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml.

Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset.

Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation.

Figure Figure
Nonmyeloablative Stem Cell Transplantation with CD8-Depleted or Unmanipulated Peripheral Blood Stem Cells: A Prospective Randomized Trial.
Authors: Willems Evelyne , Castermans Emilie , Baron Frédéric , Baudoux Etienne , Wanten Nadine , Bonnet Christophe , Hafraoui Kaoutar , Frère Pascale , Fillet Georges , Beguin Yves
Published: April 2020

Background: In a previous pilot study, we demonstrated that CD8-depletion of the graft apparently reduced the severity of AGvHD without impairing the GvL effect after peripheral blood stem cell (PBSC) transplantation with a nonmyeloablative conditioning (NMSCT).

Aim of the study: To evaluate the effect of CD8-depletion on graft rejection, AGvHD and CGvHD, and relapse.

Patients: 53 patients were randomised between CD8-depletion (group 1) (n=25) and no manipulation (group 2) (n=28). Two patients in the CD8 group were excluded for poor CD34+ cell count collected. Diagnoses were: AML (n=3), CML-AP (n=2), MDS (n=14), MPD (n=3), CLL (n=5), NHL (n=14), MM (n=8) and RCC (n=2). Median age was 57 (range 36–69) yrs. After conditioning with 2 Gy TBI with (n=39) or without (n=12) fludarabine, patients received PBSC from family (n=21) or unrelated (n=30) HLA-matched donors. CD8-depletion was carried out using the Eligix system and GvHD prophylaxis consisted in CyA and MMF.

Results: CD8 depletion removed 96% of CD8+ cells so that the number of CD8+ cells infused was 6.8 vs 136.8 x108 cells/Kg in group 2 (p<0.0001). AGvHD of any grade was observed in 13 (56%) patients in group 1 and 17 (61%) in group 2 (NS); it was grade 3–4 in 1 (4%) and 5 (18%) patients in groups 1 and 2, respectively (NS). Limited and extensive CGvHD developed in 3 and 1 patients in group 1 and in 7 and 2 patients in group 2, respectively (NS). Nine patients in group 1 and 12 in group 2 received unmanipulated DLI for poor chimerism or disease progression. Eight (3 initial and 5 late) graft failures were observed in group 1 and one (late) in group 2. Full donor chimerism was achieved in 57% (group 1) and 50% (group 2) at day 100, and in 73% (group 1) and 59% (group 2) (NS) at 1 yr. The 2-yr OS and PFS rates are 55% and 43 % in group 1 vs 59% and 46% in group 2, respectively (NS). Four (17%) patients died of their disease in group 1 vs 3 (11%) in group 2 (NS). Two patients died of severe AGvHD in group 2 vs none in group 1.

Conclusion: In vitro CD8-depletion results in higher rates of graft failure after NMSCT. The incidence of acute and chronic GvHD is not reduced but there is a trend towards reduced severity of AGvHD. Relapse and survival rates are not changed by this strategy.
Absolute Number of Transplanted CD34+ Cells Expressing c-mpl (CD110) Correlates with Speed of Platelet Engraftment Following Autologous Stem Cell Transplantation.
Authors: Sartor Mary , Garvin Frances , Antonenas Vicki , Webb Melina , Bradstock Kenneth F. , Gottlieb David J.
Published: April 2020

Recovery of neutrophil numbers after peripheral blood stem cell transplantation (PBSCT) is closely associated with graft CD34+ cell dose. Predicting the speed of platelet recovery is more difficult but would be of value given that a significant minority of patients experience delayed platelet recovery and bleeding complications after transplantation.

In this study we retrospectively analysed the graft composition of 29 patients who underwent autologous transplantation, using blood stem cells mobilized with cyclophoshamide and G-CSF, to assess the utility of c-mpl expression on CD34+ cells as a predictor of platelet engraftment (ie, time to platelet count greater than 20 x 109/L for three consecutive days without the need for platelet transfusion). Absolute CD34+ cells and CD34 subsets expressing c-mpl were enumerated using a published single platform viable CD34 flow cytometry assay (BMT, 36: 199–204,2005).

Of the 29 patients, 7 required at least 21 days for platelet engraftment. These patients received a median graft dose of 5.7 x 104 CD34+CD110+ cells/kg compared with a median dose of 13.4 x 104 cells/kg received by patients who experienced platelet engraftment within 21 days of transplant (p=0.013). In contrast, there was no difference in the number of CD34+ cells/kg infused (4.0 v 4.9 x 106/kg for > or < 21 days for platelet engraftment respectively, p=0.23). There was a poor correlation between the absolute number of CD34+ cells and the number of CD34+CD110+ cells in the graft (r2 = 0.48). Similarly there was no correlation between the percentage of CD34+ cells expressing c-mpl and the speed of platelet engraftment (8.1 v 5.8%) for > or < 21 days for platelet engraftment respectively, p=0.39). Patients with >21 days for platelet engraftment received platelet transfusions more often than those with <21 days for platelet engraftment (median 9 v 2 transfusions, p <0.001).

The absolute number of CD34+/CD110+ cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed (>21 days) platelet engraftment. Those with <6 x 104 CD34+/CD110+ cells/kg are at particularly high risk of delayed platelet engraftment, requiring multiple transfusions after transplantation.
Correlation of Clinical Response to Dasatinib (BMS-354825) with BCR-ABL Mutation Status in Imatinib- Resistant Patients (pts) with Chronic Myeloid Leukemia (CML) Treated at MD Anderson Cancer Center (MDACC).
Authors: Jabbour Elias , Cortes Jorge , Talpaz Moshe , Jones Dan , O’Brien Susan , Nicaise Claude , Kantarjian Hagop
Published: April 2020

Resistance to imatinib in CML occurs most frequently through mutations of the BCR-ABL kinase domain. Dasatinib is an orally available, dual SRC/ABL kinase inhibitor with 300-fold greater potency than imatinib, and with preclinical activity against all but one (T315I) type imatinib-resistant Bcr-Abl mutants. Dasatinib is currently used to treat pts with CML in chronic phase (CP), accelerated phase (AP), or blast phase (BP) or with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). We analyzed the response to dasatinib among pts with Bcr-Abl kinase mutations. Prior to therapy, peripheral blood samples were analyzed for mutation by DNA sequencing. 26 pts with 13 different imatinib-resistant point mutations in the Bcr-Abl kinase domain were treated (CP n=10, AP n=11, BP n=4, ALL n=1). The most common mutations were G250E/A (n=7, 27%), T315I (n=3, 12%), F317L (n=3, 12%), and E355G/A (n=3, 12%). 20 (77%) pts responded to therapy. Responses were: major molecular remission (MMR) in one pt (4%), complete cytogenetic response (CGCR) in 3 (12%), partial cytogenetic response (CGPR) in 5 (19%), complete hematologic response (CHR) in 6 (23%), partial hematologic response (PHR) in 3 (12%), and return to CP in 2 (8%). 6 pts did not respond: 3 of them were in AP (T315I, L364I, and G250E) and 3 in CP (2 T315I and F317L). Three pts (1 CP [F317L], 2 BP [M351T and E355G]) have lost their response (CGPR, CHR, and PHR) after a median of 3 months (range, 2–4 months) without developing any detectable new mutations. The median duration of response for the other 19 pts was 5+ months (range 1+–14+); 4 pts (1 CP [F486S], 3 AP [E255V and 2 G250E]) have a sustained response (1 MMR, 1 CGCR, and 2 CHR) beyond 6 months. Among 12 pts with P-loop mutations (2 in CP, 8 in AP, 2 in BP) 11 (92%) responded to therapy and their median survival since the start of therapy is 5+ months (range, 1+-11+ months). In 2 pts (CHR) subsequent analysis revealed persistence of mutations (E355G and E255V). In one pt G250E mutation occurred while patient was on treatment, but has remained in CHR. We conclude that clinical activity of dasatinib in imatinib-resistant CML is observed in pts with a wide range of imatinib-resistant Bcr-Abl kinase domain mutations. Pts harboring the T315I mutations are resistant to BMS-354825.
Correlation of Different Responses to Imatinib on Survival of Patients (pts) with Chronic Myelogenous Leukemia (CML) in Accelerated (AP) and Blast Phase (BP).
Authors: Aoki Etsuko , Kantarjian Hagop , O’Brien Susan , Rios Mary Beth , Ravandi Farhad , Verstovsek Srdan , Garcia-Manero Guillermo , Shan Jianqin , Cortes Jorge
Published: April 2020

Imatinib has remarkable activity in CML in all phases, with high rates of hematologic and cytogenetic response. However, in AP and BP responses are frequently less than complete hematologic remission (CHR), including partial hematologic remission (PHR) and return to chronic phase (second CP). The objective of this study was to investigate whether different responses to imatinib conferred a survival advantage in pts with CML in AP and BP. We evaluated 214 pts in AP and 76 pts in BP (11 lymphoid, 63 myeloid, 2 unclassified) treated with imatinib. Median age for AP pts was 49 yrs (range: 22–82 yrs) and for BP, 54 yrs (19–75 yrs). With a median follow-up of 49 months (1.9–67.5 months), 82 (38%) AP and 66 (87%) BP pts have died. A CHR and major cytogenetic (CG) response (MCR) have been achieved in 87% and 53% of AP pts, and 37% and 14% of BP pts, respectively. For AP pts the median overall survival (OS) and progression free survival (PFS) were 65 and 49 months, respectively. In BP pts, median OS and PFS were 7 and 3 months. The OS by best response to imatinib was as follows:

Response No. (%) Median OS (months) p value Median PFS (months) p value CG CR=complete CG response (Ph=0%), CG PR=partial CG response (Ph<35%), CG minor=minor CG response (Ph=35–95%), Marrow CR/PR=marrow complete/partial response Accelerated phase CG CR 96 (45) Not reached <0.0001 Not reached <0.0001 CG PR 13 (6) 43 28 CG minor 13 (6) 48 31 CHR 57 (27) 34 22 PHR 16 (7) 14 6 Resistant 11 (5) 5 3 Blast Phase CG CR 6 (8) Not reached <0.0001 Not reached <0.0001 CG PR + minor 8 (11) 12 5 CHR + PHR 12 (16) 13 11 Second CP 6 (8) 9 4 Marrow CR/PR 9 (12) 5 4 Resistant 25 (33) 4 2

We conclude that although cytogenetic responses confer the most survival advantage in AP and BP after imatinib therapy, hematologic responses are associated with a survival benefit.
Allogeneic Stem Cell Transplantation Using a Reduced Intensity Conditioning (RIC) Regimen Has the Capacity To Produce Durable Remissions and Long Term Disease-Free Survival in Patients with High Risk Acute Myeloid Leukemia (AML).
Authors: Tauro S. , Mackinnon S. , Peggs K. , Begum G. , Mahendra P. , Cook G. , Marsh J. , Milligan D. , Goldstone A. , Hunter A. , Khwaja A. , Chopra R. , Littlewood T. , Peniket A. , Parker A. , Jackson G. , Hale G. , Cook M. , Russell N. , Craddock C.
Published: April 2020

The toxicity of allogeneic stem cell transplantation (SCT) can be substantially reduced by the use of a reduced intensity conditioning (RIC) regimen and this has increased the proportion of patients with myeloid malignancies eligible for allogeneic transplantation. However, the capacity of RIC allografts to produce durable remissions in patients with AML and MDS has not yet been defined and consequently their role in the management of these diseases remains conjectural. Seventy-six patients with high risk AML or MDS were allografted using a fludarabine/melphalan RIC regimen incorporating alemtuzumab. The median age of the cohort was 52 years (range 18–71 years). There were 46 males and 30 females; 41 patients received an allograft from an unrelated donor and 35 from a matched sibling donor. The 100 day transplant related mortality was 9% and no patient developed greater than Grade 2 graft-versus-host disease (GVHD). With a median follow-up of 36 months (range 13–70 months) 27 patients were alive and in remission with a 3y actuarial overall survival (OS) and disease-free survival (DFS) rate of 41% and 37% respectively. The 3y OS and DFS of patients with AML in complete remission (CR) at the time of transplantation was 48% and 42% respectively. In a multivariate setting, Cox regression analysis demonstrated prognostic significance of disease stage at the time of transplant with improved DFS in patients with AML in CR at the time of transplantation (HR=2.03, 95% CI=1.02–4.07). Disease relapse was the commonest cause of treatment failure occurring at a median time of 6 months post-transplant. Fourteen out of 27 (52%) patients relapsed within 6 months and 23/27 (85%) within twelve months defining a relatively narrow window for therapeutic intervention to prevent disease recurrence. The extended follow-up in this series identifies a high risk of early disease relapse but provides evidence that RIC allografts are capable of producing sustained disease free survival in a significant number of patients with AML who would be ineligible for allogeneic transplantation using a myeloablative conditioning regimen.
Superior 12-yr Survival (66% vs 30%) with 5-yr Continuous (Rc) vs “Discontinuous” Remission (Rd): Results of Total Therapies 1 & 2 (TT1,2) for Multiple Myeloma (MM).
Authors: Barlogie Bart , Rasmussen Erik , Tricot Guido , Milner Teresa , Anaissie Elias , van Rhee Frits , Shaughnessy John , Epstein Joshua , Hollmig Klaus , Zangari Maurizio , Crowley John
Published: April 2020

Background: The extension of overall survival (OS) in MM has been partly linked to increasing CR rates especially with high-dose melphalan. Data bases of TT1 and TT2 were examined to determine whether 5-yr Rc was key to prolonged OS.

Patients and Methods: TT1 enrolled 231 and TT2 668 patients (median follow-up, 4 years). Pre-study characteristics of Rc vs Rd patients surviving >= 5yrs were compared. Furthermore, subsequent OS was examined in the context of pre-treatment variables, TT2 vs TT1 and Rc vs Rd. Only those Rd patients who were event-free (CR or PR) at least 1 year prior to the 5-yr landmark were included in this analysis.

Results: Among 231 TT1 + TT2 patients surviving >= 5yrs, more TT2 patients were Rc compared to Rd (60% vs 19%, p<0.001) and more older patients were Rc (at least 65: 13% vs 3%, p=0.019). Importantly, baseline LDH, B2M, CRP, albumin, presence of cytogenetic abnormalities (CA) and whether thalidomide was part of TT2 were evenly distributed among Rc and Rd groups. Furthermore, the 5-yr risk profile of Rc patients was lower than that of Rd patients: lower bone marrow plasma cell % (at least 33%: 0% vs 14%, p=0.003), higher albumin (less than 3.5g/dL: 7% vs 16%, p=0.047), higher Hb (less than 10g/dL: 4% vs 16%, p=0.023), less frequent CA13 (1% vs 11%, p=0.007). Rc patients had significantly longer subsequent survival than those with Rd (12-year survival from enrollment and 7 yrs post-landmark: 66% vs 30%, p<0.001) (Figure). In addition to Rc, univariately significant factors for post 5-year OS were data at 5 years on CA, albumin, B2M, CRP and creatinine. Rc and no CA at 5yrs were the key factors favoring long subsequent survival; thus, 12-yr OS rates decreased from 57% in the 94 patients with Rc/noCA to 44% in 44 with noRC/noCA; 8 of 9 noRc/CA patients have died by year eight; 1 of the 8 Rc/CA patients has died.

Conclusion: 5-yr Rc appears to be an important prerequisite for prolonged subsequent OS, which could be significantly increased with better therapy (TT2 vs TT1). Post-5-yr favorable survival predictors included several laboratory data at 5yrs, especially absence of CA.

Overall Survival from 5 Years Post Enrollment by Remission Status at 5 Years (Interrupted vs Continuous Remisssion) Overall Survival from 5 Years Post Enrollment by Remission Status at 5 Years (Interrupted vs Continuous Remisssion)
Total Therapy 3 (TT3) Incorporating VelcadeR (V) Intro Upfront Management of Multiple Myeloma (MM): Comparison with TT2 + Thalidomide (T).
Authors: Barlogie Bart , Tricot Guido , Rasmussen Erik , Anaissie Elias , van Rhee Frits , Zangari Maurizio , Hollmig Klaus , Epstein Joshua , Shaughnessy John , Crowley John
Published: April 2020

Background: V effects partial responses in 1/3 of patients with advanced and refractory MM and was more effective when combined with T/dexamethasone (D) in our VTD regimen (Barlogie et al. Blood2004; 103: 20). TT3 was designed to build on the success of TT2 by providing T to all patients and incorporating V in induction, consolidation and maintenance therapies to increase CR to 60% and 2-yr EFS for patients with normal/abnormal metaphase cytogenetics to 95% /75%.

Patients and Methods: TT3 consists of 2 induction cycles with VDT-PACE (PBSC collection after the first cycle), followed by melphalan 200mg/sqm-based tandem transplants with peri-transplant T+D, 2 consolidation cycles with VDT-PACE, 1 year maintenance with VTD and 2 years with T+D. Of the 162 patients enrolled prior to 4/20/2005, 156 completed induction therapy, 147 the first and 113 the second transplant. Response rates, toxicities during induction, and stem cell collection results were compared with 314 TT2+T patients.

Results: First and second transplant on TT3 were completed faster, at medians of 3 and 5 mo, compared to 5 and 10 mo, respectively, on TT2+T (p<0.001/p<0.001); 92%/78% (TT3) vs 89%/68% (TT-2+T) completed first and second transplant, respectively, (p0.24/p=0.028). The probability of achieving near-CR (n-CR: only immunofixation positive) at 12 mo was 81% with TT3 and 64% with TT2+T (p=.001). The median number of CD34/kg (x106) was 27 with TT3 vs 20 with TT2+T (p<.001). Treatment-related mortality (TRM) at 12 mo was 4% with TT3 and 6% TT2+T (p=.3). With a median follow-up of 9 mo, 18 patients have experienced an event and 14 have died (disease-related 7; treatment-related 6, other 1). Compared to event-free TT3 patients, these 18 patients were older (≥ 65 years: 50% vs 27%; p=0.04), more frequently had metaphase abnormalities (50% vs 24%; p=0.02) and had elevated LDH (50% vs 19%; p=0.003). With respect to grade 3–4 toxicities during induction, TT3 patients had fewer thrombo-embolic events, (p<.001), less febrile neutropenia (p=.03), somnolence (p=.007), sensory neuropathy (.005) and dizziness (< .001), but more anorexia (p< .001) and renal insufficiency (p=.004).

Conclusion: TT3 appears to be more effective in inducing ≥ n-CR, compared to TT-2+T, partly due to a higher percentage of patients completing the intended 2 transplants. TRM is not different. It is too early to assess the true CR rate and the 2-y EFS according to cytogenetics.
Early Serum Free Light Chain Responses Following High-Dose Melphalan and Stem Cell Transplantation for AL Amyloidosis Predict Treatment Outcomes.
Authors: Sanchorawala Vaishali , Wright Daniel G. , Quillen Karen , Fisher Catherine , Skinner Martha , Sedlin David C.
Published: April 2020

AL amyloidosis is caused by a clonal plasma cell dyscrasia and characterized by widespread, progressive deposition of amyloid fibrils derived from monoclonal Ig light chains, leading to multisystem organ failure and death. Aggressive treatment of AL amyloidosis with high-dose melphalan followed by autologous stem cell transplant (HDM/SCT) can induce hematologic and clinical remissions and extend survival. Several approaches have been used to define hematologic responses following HDM/SCT and other forms of treatment. The standard definition of a hematologic complete response (CR) that we have used requires that there be no evidence of a persistent monoclonal gammopathy by immunofixation electrophoresis (IFE) of serum and urine proteins, or of a persistent plasmacytosis or plasma cell clonality in a bone marrow biopsy by immunohistochemistry. Others have defined hematologic responses according to reductions in free light chain (FLC) measurements. Treatment responses as defined by both criteria correlate with survival and clinical improvement following HDM/SCT. We have carried out a prospective analysis of HDM/SCT treatment outcomes for patients with AL amyloidosis to determine the extent to which early FLC responses within weeks of treatment predict hematologic CR, as defined by our standard criteria. Serum free light chain concentrations (FLC) were measured by a sensitive nephelometric immunoassay in 31 patients with AL amyloidosis, between 2003–2005, 1–3 weeks after treatment with HDM/SCT. Hematologic responses, as defined by standard criteria, as well as FLC responses were subsequently determined at 3, 6 and 12 months. Serum FLC levels or κ/λ FLC ratios were abnormal and informative in 28 patients (90%) prior to HDM/SCT, and these patients were included in subsequent analyses. Twenty patients (71%) achieved normalization of abnormal serum FLC levels or ratios within 1–3 weeks of undergoing HDM/SCT. Of these 20 patients, 13 patients (65%) subsequently achieved a hematologic CR as defined by standard criteria, while 7 (35%) did not, within 3 months following HDM/SCT. In contrast, none of the 8 patients with no demonstrable FLC response within 1–3 weeks of HDM/SCT, were found to have achieved a hematologic CR subsequently. In conclusion, meaningful quantitative FLC responses (or lack of response) can be detected within weeks following HDM/SCT treatment that predict hematologic responses, as defined subsequently by standard criteria based on IFE and marrow studies (p=0.0018 by chi square analysis). Moreover, a lack of an early FLC response predicts for hematologic non-CR. We anticipate that prospective studies of FLC responses in HDM/SCT and other clinical trials for AL amyloidosis will eventually lead to more rapid assessment of treatment responses that will guide therapeutic decisions.
Prognosis in Multiple Myeloma (MM): Characterization of “Losers” (L) in Good Risk and “Winners” (W) in High Risk Disease.
Authors: Anaissie Elias , Rasmussen Erik , Shaughnessy John , Tricot Guido , Barlogie Bart
Published: April 2020

Background: The clinical course of patients with MM is highly variable and, among currently available standard features, the presence of metaphase cytogenetic abnormalities (CA) is the parameter with the single best discriminatory power. The aim of this investigation was to determine, among the 1/3 high-risk patients with CA the W and among the remaining low-risk patients the L.

Patients and Methods: We evaluated the baseline characteristics of > 2500 patients who received at least 1 high-dose melphalan-based autotransplant (AT). Poor risk was defined as pre-AT CA (within 90 days) while good risk was defined as no pre-TX1 CA. Poor outcome was death within 2 years of AT while good outcome was survival for ≥ 5 years. Four groups were identified: 403 patients without CA and >5yr survival (noCA/W), 288 without CA and <2yr survival (noCA/L), 122 with CA surviving >5yr (CA/W) and 350 with CA surviving <2yr (CA/L).

Results: Among both CA and no CA groups, univariately significant pre-AT differentiating features between W vs L were serum albumin > 3mg/dL, b2M<3 mg/dL, CRP<6 mg/dL, hemoglobin > 10 mg/dL and platelet counts > 100/μL, LDH < 190 mg/dL, age < 65, female gender and primary treatment on Total Therapy protocol. By multivariate analysis, independently highly significant pre-AT factors that distinguished the 5 yr survivors from the early death (<2 years) patients included primary treatment on Total Therapy protocol, serum albumin > 3mg/dL, hemoglobin > 10 mg/dL, B2M<3 mg/dL, platelet counts >100/μL and CRP<6 mg/dL. These independent prognostic factors were almost identical for CA and noCA patients except for Hgb which was slightly more important than albumin in the CA population.

Conclusion: Among MM patients receiving AT, treatment on Total Therapy protocol, higher Pre-AT albumin, hemoglobin and platelet count and lower b2M and CRP confer a survival advantage regardless of cytogenetic status (CA or noCA) and distinguish the W among the high-risk disease from the L among the low-risk patients.
Second Autologous Stem Cell Transplant (ASCT) as Salvage Therapy for Relapsed Multiple Myeloma.
Authors: Olin Rebecca L. , Porter David L. , Luger Selina M. , Schuster Stephen J. , Tsai Donald , Nasta Sunita , Perl Alexander , Loren Alison W. , Goldstein Steven , Glatstein Eli , Siegel Donald L. , Vogl Dan T. , Mangan Patricia , Cunningham Kathleen , Stadtmauer Edward A.
Published: April 2020

Introduction: Autologous stem cell transplant (ASCT) as part of initial therapy has been shown to prolong survival of patients with multiple myeloma, with some achieving durable complete remission. However, the majority of patients ultimately relapse after ASCT and require salvage treatment. Options for the treatment of such patients have increased significantly over recent years, including not only novel chemotherapeutic and biological agents but also additional ASCTs. We performed a retrospective analysis of our experience with salvage ASCT for multiple myeloma to determine which clinical variables influence outcome.

Methods: Between October 1992 and February 2005, we performed 342 ASCTs for multiple myeloma. Twenty-six of these were salvage transplants for relapsed disease after prior ASCT, and all were included in the analysis. Patients who received two planned (tandem) ASCTs were not included.

Results: The median age at diagnosis was 47 (range 25–66), and median ISS and DS stages at diagnosis were 1 and 2, respectively. The initial ASCT was melphalan-based in 21/26; six (23%) achieved a complete response (CR) to the initial transplant, and fifteen (58%) achieved a partial response (PR). The median event-free survival (EFS) after the initial transplant was 19.5 months (range 2–60). The median time between initial and salvage ASCT was 2.6 years (range 0.3–7.6). Twenty-two patients (85%) received non-transplant therapy between ASCTs, and the median number of lines of therapy prior to salvage ASCT was 3. At the time of salvage ASCT, the median age was 52.5 (range 28–69). Fourteen patients received melphalan alone, eight received melphalan/TBI, and four received other regimens. Eleven patients (42%) achieved a response to therapy (1 CR, 10 PR). One patient (4%) died of transplant-related toxicity. The median follow-up after salvage ASCT is 12 months (range 0.2–58). Median EFS is 9 months, and median overall survival (OS) is 36 months. The 2-year EFS is 14%, and 2-year OS is 52%. On univariate analysis, both response to and EFS after initial transplant significantly predict improved EFS after salvage transplant (p=0.0008 and p=0.0065 respectively). Both also predict improved OS (p=0.03 and 0.0005 respectively). A greater than 12 month interval between first and second transplant also correlated with OS (p=0.04). There was no significant difference in EFS or OS by preparative regimen. Interestingly, type of response to the salvage transplant (CR/PR or less than PR) did not predict improved EFS or OS.

Conclusion: This study suggests that salvage ASCT after relapse from initial ASCT is a feasible therapy for patients with heavily treated multiple myeloma, particularly those with a prolonged response to the first transplant.
Synergistic Induction of T Cell Acute Lymphoblastic Leukemia by Functionally Distinct Mutations in NOTCH1.
Authors: Chiang Mark Y. , Hong Sai , Shestova Olga , Lee Woojoong , Xu Mina , Pear Warren S. , Aster Jon C.
Published: April 2020

Recent observations that Notch1 mutations are present in more than 50% of human T-ALL patient samples indicate that Notch signals have a central role in the pathogenesis of T-ALL (Weng et al., 2004, Science, 306, 269–271). Notch1 mutations are of two types: i) mis-sense mutations in an extracellular heterodimerization (HD) domain; and ii) frameshift or stop codon mutations that result in deletion of an intracellular PEST destruction box. The likely effect(s) of these mutations is to i) cause heightened proteolytic activation of Notch1, an event that requires the enzyme gamma-secretase; and to ii) increase the duration of Notch1 signaling, respectively. To investigate the contribution of the HD and PEST domain mutations to the pathogenesis of human T-ALL, Notch1 alleles with common HD mutations were introduced retrovirally into hematopoietic progenitor cells, which were then transplanted into lethally irradiated mice. These mice developed transiently circulating abnormal CD4+CD8+ T cells, but failed to develop leukemia after 9 months of monitoring. Similar findings occurred when Notch1 alleles bearing only PEST mutations were introduced into mice, as previously noted by others (Feldman et al., 2000, Blood, 96, 1906–1913). However, when HD mutations were combined with a PEST mutation in cis, the mice developed persistent circulating CD4+CD8+ T cells, leukocytosis, and eventually T-ALL (median time, 18 weeks post-transplantation). These tumors infiltrated lymphoid organs, marrow, and viscera, were monoclonal or oligoclonal, and had intact proviral integrations. Cell lines derived from these tumors are susceptible to gamma-secretase inhibitor-mediated cell death, which indicates a continued reliance on Notch signaling for growth and survival. These results show the utility of the murine BMT assay to score Notch1 mutants found in human T-ALL with respect to leukemogenicity, and suggest that the level of signal that is necessary to induce and sustain Notch1-associated T-ALL is relatively high. It will be of interest to determine if HD domain mutations generally require PEST deletions in cis to induce T-ALL, or if weak and strong mutations of varying leukemogenic potential exist.
Expression of ATRA Inducible RARα2 Isoform Is Deregulated in AML.
Authors: Glasow Annegret , Barrett Angela , Gupta Rajeev , Grimwade David , von Lindern Marieke , Enver Tariq , Zelent Arthur
Published: April 2020

Retinoids exert a variety of effects on both normal and malignant hematopoietic cells. To date, three different retinoic acid receptor (RAR) and retinoid X receptor (RXR) genes have been characterized, each encoding multiple N-terminal protein isoforms. RXRs serve as co-regulators for RARs, and many other nuclear receptors integrating different signalling pathways. All-trans-retinoic acid (ATRA) signaling pathway is of critical importance for optimal myelomonocytic differentiation and its disruption by translocations of the RARα gene leads to acute promyelocytic leukemia (APL). APL associated fusion oncoproteins, such as PML-RARα and PLZF-RARα, function through recruitment of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), thus promoting an inactive chromatin state and leading to repression of RARα target genes. Recently, we demonstrated that up-regulation of RARα2 expression by ATRA directly correlates with differentiation of APL and non-APL AML cells and that RARα2 transcription is silenced by DNA methylation in AML cell lines. Using primary AML samples as well as normal cord and peripheral blood derived cells representing different stages of myelomonocytic development we now show that expression of RARα2 increases with maturation of hematopietic cells. Expression of RARα1 on the other hand, which is transcribed from a distinct promoter, remains relatively constant throughout the different stages of myelomonocytic development. The levels of RARα1 expression in various primary AML cell types appear to be similar to those found in normal hematopietic cells. Consistent with data derived from AML cell lines, however, the RARα2 isoform is poorly expressed in all samples. Compared with CD34+/CD133+ or CD34+ progenitors, and more mature CD33+ myeloid cells, RARα2 is expressed at much lower levels in a variety of primary AML cells and its expression is not effectively induced by myelomonocytic growth factors and/or ATRA. Negatively acting epigenetic changes, such as DNA methylation, appear to be responsible for deregulated expression of RARα2 in AML cells, although their pattern and extent differs significantly between AML cell lines and primary AML samples. Taken together our data suggest that agents, which revert negatively acting epigenetic changes may restore expression of the RARα2 isoform in AML cells and render them more responsive to ATRA as well as other differentiation inducers.
MHC Class-II Expression Reveals Differential Regulation of BLIMP-1 Target Genes.
Authors: Tooze Reuben , Stephenson Sophie , Doody Gina , Rawstron Andy
Published: April 2020

BLIMP-1 initiates plasma cell differentiation by inhibiting the expression of a limited set of transcription factor genes. BLIMP-1 mediates repression of its targets by promoter occupancy and recruitment of the Groucho corepressor, histone deacetylase and the G9a lysine methyltransferase. The relative contribution of these mechanisms to the stable silencing of BLIMP-1 target genes in plasma cells is not known. Epigenetic changes initiated by BLIMP-1 could be maintained by independent factors or alternatively may be dependent on continuous occupancy of the promoter by BLIMP-1. The aim of this study was to investigate the mechanisms operating at two well-defined BLIMP-1 target genes, MHC2TA and PAX-5. Repression of PAX-5 is essential for extinction of the B-cell phenotype, while repression of MHC2TA mediates the loss of MHC class-II expression characteristic of plasma cell differentiation. PAX-5 is silenced in virtually all cases of myeloma, whereas MHC class-II continues to be expressed in a subset of primary myeloma cells and many myeloma cell lines. Using chromatin immunoprecipitation we demonstrate that BLIMP-1 constitutively binds MHC2TA promoter-3 and is present equally in MHC2TA expressing and non-expressing myeloma cell lines. In expressing cells MHC2TA promoter-3 is associated with acetylation of histone H3 lysine-9, a marker of open chromatin, whereas histone H3 lysine-9 is neither acetylated nor methylated in non-expressing cells. In contrast the PAX-5 promoter is associated with trimethylation of histone H3 lysine-9, a marker of repressed heterochromatin, in all the cell lines examined, but constitutive BLIMP-1 occupancy is not detectable. Although BLIMP-1 does not silence MHC2TA expression in the MHC class-II positive U266 cell line, it is mediating transcriptional repression, since siRNA knockdown of BLIMP-1 is associated with elevated MHC2TA mRNA and MHC class-II surface expression. In contrast PAX-5 mRNA and its target CD19 continue to be repressed. MHC class-II re-expression was associated with loss of CD138 and the entry of the majority of cells into apoptosis. Our data demonstrate that MHC class-II expression in myeloma cell lines is associated with defective silencing of MHC2TA despite BLIMP-1 occupancy, and reveal differences in the contribution of epigenetic modifications and promoter occupancy in the maintenance of target gene silencing by BLIMP-1. Loss of BLIMP-1 results in a partial reversion of plasma cell phenotype that is associated with induction of apoptosis. As continual high levels of BLIMP-1 expression appear to be critical for plasma cell survival, suppression of BLIMP-1 represents a potential therapeutic pathway.
Detection of the Acquired Tyrosine Kinase JAK2 Gene Mutation by Real Time PCR as Minimal Residual Disease Marker for Patients after Allogeneic Hematopoetic Stem Cell Transplantation.
Authors: Elmaagacli Ahmet H. , Steckel Nina K. , Koldehoff Michael , Ditschkowski Markus , Trenschel Rudolf , Beelen Dietrich W.
Published: April 2020

Recently, a single point mutation (VAL617Phe) in the tyrosine kinase gene JAK2 was identified in some patients with polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis by Baxter and coworker. Here, we assessed the frequency of these acquired JAK2 gene mutations by real-time PCR in patients with myelofibrosis with myeloid metaplasia (MMM) (n=25), atypical bcr-abl negative CML (n=8), MDS (n=53), AML M6 (erythroleukaemia) (n=8) and AML megakaryocytic leukaemia) M7 (n=2) prior to a scheduled allogeneic transplant. Further, we evaluated the use of the detection of JAK2 gene mutation as a MRD marker after transplant. A JAK2 gene mutation was found in 13 of 25 patients with MMM, 1 of 8 patients with atypical CML, 2 of 2 patients with AML M7, but none in patients with MDS (n=53) or patients with AML M6 (n=8). All tested 24 healthy donors had no detectable JAK2 mutation by PCR. In 15 patients with a detectable JAK2 gene mutation prior to transplant, we evaluated the chimerism status and the detection of the JAK2 mutation by PCR simultaneously. Four of these 15 patients revealed a mixed chimerism after transplant at different time periods. In three of these four patients a JAK2 gene mutation was detectable contemporarily by PCR, whereas 11 patients with a complete chimerism detected by STR-PCR had no evidence for a JAK2 mutation at the same time point. Three patients with MMM in whom a JAK2 mutation could be detected again relapsed again of MMM. These data demonstrate that JAK2 gene mutation can be used as a MRD marker for MMM in patients who had this gene mutation prior to transplant.
BCR-ABL Decreases the Expression of CCN3 Leading to Increased Clonogenic Potential and Cell Growth.
Authors: McCallum Lynn M.R. , Lu Wanhua , Price Susan , Planque Nathalie , Perbal Bernard , Pierce Andrew , Whetton Anthony , Irvine Alexandra E.
Published: April 2020

Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we have identified downregulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity and detected reduced CCN3 expression in human CML cell lines and primary human CML cells. We now report a reciprocal relationship of BCR-ABL and CCN3 expression and the functional consequence of expressing CCN3 in BCR-ABL+ cells. Real-time PCR was used to examine the relationship between BCR-ABL and CCN3 expression in human K562 cells. Parental K562 cells showed high expression of BCR-ABL (4.68 x104 transcripts in 5 μL of cDNA) whilst CCN3 expression was not detected. Treatment with siRNA directed against BCR-ABL (0.5 μg per106 cells) for 72 hours resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (mean Ct change 1.9 ± 0.2 and 2.6 ± 0.5 for BCR-ABL and CCN3 respectively, n=3, p=0.001). Similarly, K562 cells treated with imatinib (1 micromolar) for 96 hours showed a 5.9 fold decrease in BCR-ABL expression and a 4.2 fold increase in CCN3 expression (mean Ct change 2.5 ± 0.1 and 2.1± 0.2 for BCR-ABL and CCN3 respectively, n=3, p=0.001). To investigate CCN3 function, we expressed CCN3 in BCR-ABL+ cells using Nucleofector technology (Amaxa, GmbH). K562 cells were transfected with either the pCb6+ vector (Invitrogen,UK) or pCb6+ vector containing the CCN3 construct. Cells were analysed 24 hours post-transfection by flow cytometry and also after 7 days in methyl cellulose culture to determine clonogenicity. Cell cycle analysis was performed on 20,000 events using the winMDI software. CCN3 expression in BCR-ABL+ cells resulted in an accumulation of cells in the subG0 phase of the cell cycle (mean for subG0 9.9% ± 4.6 and 21.8% ± 0.7 for the pCb6+ vector alone and pCb6+ vector containing CCN3 construct respectively). CCN3 expression significantly increased the number of cells within the subG0 area of the cell cycle (n=3, p=0.027). In addition, CCN3 expression reduced the clonogenic capacity of BCR-ABL+ cells. K562 cells transfected with the pCb6+ vector containing CCN3 construct formed significantly fewer colonies on methyl cellulose in comparison to cells that had been transfected with the pCb6+ vector alone (n=3, p=0.027).

This study demonstrates a reciprocal relationship between CCN3 and BCR-ABL expression. CCN3 is known to be a negative growth regulator and increased expression of CCN3 in BCR-ABL+ cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.