bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the Sequential High-Dose Chemotherapy Followed by Autologous Stem Cell Support with and without Rituximab in Relapsed and Refractory Aggressive Non-Hodgkin’s Lymphoma.
Authors: Sieniawski Michal , Staak Jan Oliver , Scheuss Helena , Glossmann Jan Peter , Diehl Volker , Engert Andreas , Josting Andreas
Published: October 2019

Introduction: Combination chemotherapy can cure patients (pts) with Non-Hodgkin’s lymphoma (NHL), but those with relapse still have a poor prognosis. High-dose chemotherapy (HDCT) with autologous stem cell support (ASCT) can improve the outcome of these pts. Rituximab demonstrated encouraging activity in aggressive NHL and showed low toxicity in the setting of combined immunochemotherapy. To investigate the influence of addition of rituximab to the intensified salvage program followed by a final myeloablative course with stem cell reinfusion we compared the group of patients treated with this program - immuntherapy group (IT) with the standard therapy group (ST) of patients treated with the same program without rituximab.

Patients and methods: Eligibility criteria for both groups were as follows: age 18–67 years, eligibility for HDCT, histologically proven CD20+ relapsed or progressive NHL. Treatment program starts with two cycles DHAP (dexamethasone, cytarabine, cisplatin); pts with PR or CR receive cyclophosphamide (4g/m2) followed by PBSC harvest; methotrexate 8g/m2 and vincristine 1,4mg/m2; and etoposide 2g/m2. The final myeloablative course is BEAM followed by ASCT. Pts in IT received additionally rituximab (375mg/m²) to the each chemotherapy cycle.

Results: In the immuntherapy and standard therapy group were enrolled 23 and 57 pts, respectively. There were 18 (78%) IT and 34 (60%) ST pts with relapsed and 5 (22%) IT and 23 (40%) ST pts with refractory disease. The majority of pts in both groups received CHOP-like regimens as first line treatment (91% IT vs. 79% ST). The response rate at the final evaluation for all patients was in IT 61% (52% CR and 9% PR) and in ST 43% (32% CR and 11% PR). The overall response rate (OR) for patients who responded to 2 cycles DHAP was in IT 83% (71% CR and 12% PR) and in ST 59% (44% CR and 15% PR). The therapy toxicity was tolerable and comparable in both groups.

Conclusion: The preliminary results suggest better therapy outcome in patient with sequential high-dose chemotherapy with rituximab compared to pts treated with the same therapy regimen without additional antibody. The combination regimen allows effective mobilization of stem cells. The tolerability of the final myeloablative BEAM was in both groups not affected by rapid sequential administration of DHAP and high doses of cyclophosphamide, methotrexate, etoposide and rituximab. The toxicity was tolerable in both groups. The full results of match pair and multivariate analysis as well as OS and FF2F will be presented.
Holmium-166 (166Ho)-DOTMP Skeletal Targeted Radiotherapy (STR™) with Melphalan and Autologous Peripheral Stem Cell Transplant (PBSCT) for Multiple Myeloma (MM).
Authors: Goodman Mark , Bensinger William I. , Giralt Sergio , Salzman Donna , Ruffner Katherine L. , Breitz Hazel B.
Published: October 2019

Background: 166Ho-DOTMP is a beta-emitting radiophosphonate that localizes specifically to the bone surfaces and can deliver high dose radiation both to the bone and bone marrow. Follow-up data from 3 clinical trials with STR as conditioning for patients with MM undergoing autologous PBSCT are presented.

Methods: In 2 Phase I/II dose-escalation trials, 83 patients received a dose of 166Ho-DOTMP STR calculated to deliver 20, 30, or 40 Gy to the red marrow; 82 pts received melphalan (140 or 200 mg/m2) ± 8 Gy TBI (n=25), followed by PBSCT. As of June, 2004, 77 subjects have been followed for at least 48 months. In a separate Phase II dosimetry trial, 12 patients received two 30 mCi tracer doses of 166Ho-DOTMP STR to determine the reproducibility of biodistribution and pharmacokinetics (PK). All pts received a 25 Gy therapy dose with concurrent IV hydration and continuous bladder irrigation, followed by 200 mg/m2 melphalan and PBSCT. These patients have been followed for at least 18 months.

Results: Up to 2.3 Ci/m2, 166Ho-DOTMP STR was given in the Phase I/II trials; 29/83 (35%) patients achieved complete response (CR) and overall response rate (CR + PR) was 64% (7 pts not evaluable). The Kaplan-Meier estimate of median survival is 5.2 years for all 83 patients. In patients who are at least 4 years post transplant who achieved a CR, the survival is 74% (n=27). In patients who achieved less than a CR at least 4 years ago, the survival is 34% (n=44). Dose-related radiation-induced kidney toxicity presented in some patients more than 6 months post-therapy. The dose of 166Ho-DOTMP STR in the Phase II dosimetry trial was 550 to 860 mCi/m2, 166Ho-DOTMP. Currently, 18 months of follow-up reveals no occurrence of hemorrhagic cystitis or > Grade 2 elevated creatinine. A CR rate of 17% with an overall survival of 92%, was observed. In 10 patients who received 166Ho-DOTMP STR 750 mCi/m2 ± 10% in the Phase I/II trial, the CR rate was 40%, and the 4-year survival was 70%. Monitoring for safety and duration of response is ongoing in all 3 trials.

Conclusion: Follow-up from the Phase I/II trials confirms that 166Ho-DOTMP STR provides favorable efficacy and safety as part of the conditioning regimen for patients with MM undergoing PBSCT. A Phase III, randomized multicenter study is now open to enrollment, comparing the safety and efficacy of 166Ho-DOTMP STR plus melphalan to melphalan alone as conditioning for PBSCT in subjects with primary refractory MM who have failed to respond to induction therapy, including high-dose dexamethasone, and are within 18 months of diagnosis.
Recovery of Polyclonal Immunoglobulin Synthesis after Autologous Stem Cell Transplantation in Multiple Myeloma Patients as a Prognostic Factor for Event-Free Survival.
Authors: Höfer Annette , Benner Axel , Klaus Jens , Breitkreutz Iris , Herrmann Doris , Cremer Friedrich W. , Gerull Sabine , Moehler Thomas , Egerer Gerlinde , Hegenbart Ute , Ho Anthony D. , Goldschmidt Hartmut
Published: October 2019

Suppression of polyclonal immunoglobulin (Ig) synthesis is one feature of multiple myeloma (MM). To evaluate if recovery of polyclonal Ig synthesis after autologous stem cell transplantation (ABSCT) influences prognosis, we have retrospectively analyzed the prognostic value of clinical and laboratory variables of 348 multiple myeloma patients who underwent their first autologous stem cell transplantation between 06.1992 and 10.2002 at the University Hospital of Heidelberg. The median follow-up was 34 months (range, 3 to 136 months). The median number of chemotherapy courses prior to first ABSCT was 5 (range, 3 to 30). Twenty-two of 348 patients underwent altogether three ABSCTs (including 18 patients with tandem transplantations), 141 had two ABSCTs (including 87 tandem transplantations) and 29 patients were treated with allogeneic transplantation as second line treatment. Recovery of polyclonal Ig synthesis was studied by serumelectrophoresis on day 100 after first ABSCT (±30 days). Full polyclonal Ig recovery was observed in 93 of 348 patients (28%), partial recovery (reduced polyclonal Ig, no monoclonal Ig) in 58 (17%), no recovery (almost no polyclonal Ig and no monoclonal Ig) in 16 (5%), polyclonal Ig recovery associated with monoclonal Ig in 95 (27%) and monoclonal Ig without polyclonal Ig in 72 patients (21%) (data missing in 14 patients (4%)). On multivariate analysis (333 patients), using Cox proportional hazards regression model stratified with respect to tandem transplantation, superior event-free survival was observed with recovery of polyclonal Ig synthesis, low beta2-microglobulin at diagnosis (<2.5mg/L) and a high haemoglobin at diagnosis, whereas MM type IgA and a high number of chemotherapy courses prior to first ABSCT were identified as adverse prognostic factors. Regarding polyclonal recovery, patients with polyclonal Ig recovery associated with monoclonal Ig had a superior event-free survival compared to patients with only monoclonal Ig without any polyclonal Ig recovery. Overall survival was superior in patients with a low β2-microglobulin at diagnosis (<2.5mg/L) and a high haemoglobin at diagnosis, and worse in patients with a high number of chemotherapy courses prior to first ABSCT. We conclude that any recovery of polyclonal Ig synthesis even when associated with monoclonal Ig, improves event-free survival, and therefore can be used as a predictor of prognosis in the follow-up of multiple myeloma patients who underwent ABSCT.
Serum Free Light Chain Responses after High-Dose Intravenous Melphalan and Autologous Stem Cell Transplantation for AL (Primary) Amyloidosis.
Authors: Sanchorawala Vaishali , Wright Daniel G. , Magnani Barbarajean , Skinner Martha , Seldin David C.
Published: October 2019

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 of plasma cell dyscrasias underlying AL amyloidosis following HDM/SCT and other forms of treatment. The 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 persistent plasmacytosis or plasma cell clonality in a bone marrow biopsy by immunohistochemistry. Others have defined hematologic response as a ≥ 50% reduction in free light chain (FLC) measurements. Hematologic responses by both criteria correlate with survival and clinical improvement following HDM/SCT. We have carried out a retrospective analysis of HDM/SCT treatment outcomes for patients with AL amyloidosis to determine the extent to which hematologic CR, by our standard criteria, correlates with FLC response. Serum free light chain concentrations (FLC) were measured by a sensitive nephelometric immunoassay in 67 patients with AL amyloidosis before and after treatment with HDM/SCT. After treatment with HDM/SCT, 27 patients (40%) achieved a CR by standard criteria. Of these 27 patients, 63% (n=17) demonstrated normalization of FLC levels and an improvement of ≥50% in FLC occurred in 100%. Of the 40 patients who did not achieve a CR, 25% (n=10) experienced normalization of FLC levels, and an improvement of ≥50% occurred in 78% (n=31), while only 5 patients (13%) experienced no significant change in FLC. The average improvement in FLC was 94% for patients who achieved a CR by standard criteria and 72% for those who did not (p=0.0001, t-test). Thus, HDM/SCT was found to induce improvements in FLC levels of ≥50% in the vast majority of AL amyloidosis patients treated with HDM/SCT (87%, or 58/67). These data indicate that a decrease in FLC of ≥50% is a substantially less stringent indicator of hematologic response than is CR, as defined by standard criteria. Nonetheless, these measures of hematologic response are complementary, since decreases in FLC can be detected earlier following treatment than changes in IFE and marrow studies required to determine CR.
CD56 as Prognostic Factor in Patients with Multiple Myeloma Treated by High-Dose Chemotherapy and Autologous Stem Cell Transplantation.
Authors: Hundemer Michael , Klein Ulrike , Hose Dirk , Cremer Friedrich , Raab Marc , Moos Marion , Ho Anthony D. , Goldschmidt Hartmut
Published: October 2019

Neuronal cell adhesion molecule (NCAM, CD56) is expressed by approximately 75% of multiple myeloma (MM) clones. The lack of CD56 expression on MM cells is described to be associated with a higher frequency of extramedullary involvement (Pellat-Deceunynck et al. 1998) and a lower overall survival in patients treated by conventional chemotherapy (Sahara et al. 2002). We analyzed the CD56 expression of myeloma cells by flowcytometry in 52 newly diagnosed patients to determine its prognostic and clinical relevance. Seventeen of 52 patients were NCAM-negative and 35 patients NCAM positive. The CD56 expression did not correlate with elevated beta2-microglobulin levels or deletions of chromosome 13q14 as investigated by interphase FISH. All patients were scheduled for treatment according to the GMMG-HD3 protocol and received 3 cycles of VAD or TAD induction chemotherapy, followed by CAD chemotherapy, stem cell collection and high dose chemotherapy with melphalan and autologous stem cell transplantation (ASCT). We analyzed the response rate (EBMT/IBMTR criteria) 3 months after the first ASCT and the event free survival (EFS) (median 479 days, range 29–911 days). Three months after the first ASCT, 6/17 of the CD56 negative patients were in CR, 7/17 in PR, 1/17 in MR and 3/17 had PD. Of the CD56 positive patients evaluated for clinical outcome 5/35 showed CR, 20/35 a PR and 10/35 a PD. In an intention-to-treat analysis, EFS showed no significant difference between these two groups. With only 13 events out of 52 cases, a longer follow up in terms of EFS and OS is required. We conclude that in patients treated by high-dose melphalan and autologous stem cell transplantation, the lack of CD56 did not correlate with a poor response rate 3 month after transplantation. Our preliminary results indicate that high-dose therapy followed by ABSCT is able to overcome the poor prognosis of CD56 negative patients treated with conventional chemotherapy.
Genoidentical Donor Versus A, B, Cw, DR, DQ, HLA Allelic-Matched Unrelated Donor for Stem Cell Transplantation in Patients with Standard Risk Haematological Malignancy.
Authors: Yakoub-Agha Ibrahim , Boiron Jean-Michel , Michallet Mauricette , Milpied Noel , Bourhis Jean-Henri , Cahn Jean-Yves , Buzyn Agnes , Gratecos Nicole , Esperou Helene , Vernant Jean-Paul , Raffoux Colette , Golmard Jean-Louis , Jouet Jean-Pierre , Kuentz Mathieu
Published: October 2019

To compare outcome of pts with standard risk hematological malignancy undergoing allogeneic stem cell transplantation (allo-SCT) from either allelic A, B, Cw, DR and DQ HLA-matched unrelated (so called 10/10) or HLA-matched sibling donor, we conducted a prospective study including 12 French BMT centres. Between 01/2000 and 12/2002, 236 consecutive pts were enrolled. Fifty five pts received unrelated allo-SCT (pheno-group) and 181 received genoidentical allo-SCT (geno-group). Conditioning regimen associated TBI and Cy in all pts. Pts who received ATG with conditioning were excluded. GVHD prophylaxis consisted on Cs-A plus MTX. Diagnoses of the underlying disease were, AL (n=175) of whom 47 were in CR > 1; CML (n=43) of whom 4 were with accelerated phase and myelodysplastic syndrome (n=18) of whom 11 with untreated disease. The analyses were performed at the reference date of december 31th 2003. The two groups were comparable in terms of patients’ initial characteristics. However, pts of pheno-group were slightly younger and had more often CML whereas those belonging to geno-group had more often acute leukemia in 1st remission . At the date of analysis, the median follow-up was 955d (373–1398). The overall survival was 63% and 52,5% (p=NS) for geno and pheno-group, respectively. Furthermore, the statistical power was insufficient to demonstrate an equivalence of the two survival curves.. Although, the occurrence of acute GVHD of grade ≥2 was statistically higher in pheno-group (67% vs 52%, p=0.0), there was no significant difference en term of TRM between the two groups (27% vs 33% for geno and pheno-group, respectively). Relapse rates were 20% vs 26% and ultimately EFS (56% vs 52%) for geno and pheno-group. In multivariable analysis, only age (>36 years) and positive-CMV serology of recipient were found to adversely influence EFS. Finally, there was no demonstration of an independent prognostic value of the donor origin on outcome. In conclusion, although, the equivalence was not fully demonstrated between the two groups, these preliminary results of allo-SCT from 10/10 HLA-matched unrelated donor compare favourably to those obtained with HLA-matched sibling donor in patients with standard risk haematological malignancies.
Impact of Molecular HLA Matching for Allogeneic Stem Cell Transplantation from Unrelated Donors.
Authors: Chalandon Yves , Halter Joerg , Guengoer Tayfun , Tichelli Andre , de Faveri Grazia Nicoloso , Chapuis Bernard , Seger Reinhard , Schanz Urs , Gratwohl Alois , Roosnek Eddy , Tiercy Jean-Marie , Passweg Jakob R.
Published: October 2019

Two hundred and fourteen unrelated donor transplants (HSCT) using peripheral blood (N=60) or marrow (N=154) as a stem cell source have been performed in Switzerland since 1990. All transplants were coordinated by the Swiss bone marrow donor registry and were performed at 4 different institutions. Median age was 28 (1–61) years, and 132 (62%) patients were male. Twenty-four patients with malignancy underwent transplants with reduced intensity conditioning regimens. T-cell depletion was performed in 45 patients. Patients had CML (62), AML (53), ALL (36), MDS/MPS (31), lymphoid malignancies (14), acquired or congenital aplastic anemia (11), or other inherited disorders (7). Sixty-two patients with AML, ALL or CML were transplanted at an early disease stage, i.e. CR1 or 1st chronic phase, the remainder with more advanced disease. All donor/recipient pairs underwent high resolution HLA typing in the LNRH, the Swiss national reference laboratory for histocompatibility in Geneva. Donor/recipient pairs were HLA-A, -B, -C, -DRB1/B3/B5, -DQB1 matched (130), or were mismatched at the HLA-A or B (10), HLA-C (29) HLA-DR (14), HLA-DQ (16) or at multiple (15) loci. Median follow-up of surviving patients was 34 months. Survival probabilities (± 95% CI) at 2 years were, in decreasing order 62 ± 9% for recipients of HLA 10/10 matched transplants, 46 ± 26% with DQB1 mismatch, 42 ± 20% with HLA-C mismatch, 30 ± 27% with HLA-DRB1 or DRB3 mismatch, 15 ± 26% with HLA-A or B mismatch and 13 ± 18% with multiple mismatches, p<0.0001. In multivariate analysis HLA compatibility, as defined by molecular typing, was the variable most significantly associated with survival and treatment-related mortality.

Multivariate analysis for survival

Factor RR (95% CI) P Age 1.014 (1.001-1.028) 0.04 HLA matching Match 1 0.001 DQ mismatch 1.38 (0.65-2,92) C mismatch 2.12 (1.04-4.32) DR mismatch 2.17 (1.21-3.89) A/B mismatch 3.66 (1.79-7.49) Multiple mismatches 4.35 (2.32-8.14)

Unrelated donor transplantation is increasingly used in Switzerland to treat a variety of disorders. Long term survival in recipients of transplants from HLA-A/B/C/DR/DQ-matched donors (10/10) is comparable to that achieved with genotypically identical sibling donors. Single mismatches at HLA-C or DQB1 loci may be more acceptable than A, B or DR-incompatibilities which are associated with decreased survival. Without a suitable donor, alternative methods to unrelated donor HSCT should be considered.
Comparison between Anti-Thymocyte Globulin and Alemtuzumab and the Possible Impact of KIR-Ligand Mismatch in Melphalan/Fludarabine Dose-Reduced Conditioning Followed by HLA-Matched and Mismatched Unrelated Stem Cell Transplantation in Patients with Multiple Myeloma.
Authors: Kroeger Nicolaus , Shaw Brownen , Iacobelli Simona , Zabelina Tatjana , Peggs Karl , Shimoni Avichai , Nagler Arnon , Binder Thomas , Schwerdtfeger Rainer , Kiehl Michael , Sayer Herbert G. , Ayuk Francis , Eiermann Thomas , Marks David
Published: October 2019

We compared anti-thymocyte globulin (ATG-Fresenius median dose 60 mg/kg: n= 48) with alemtuzumab (Campath-1H 100mg: n=25) in 73 patients with multiple myeloma, who underwent dose-reduced conditioning with melphalan and fludarabine, followed by allogeneic stem cell transplantation from matched (n=63) or mismatched (n=10) unrelated donors. Patients of the ATG group had higher age (median 50 vs 47 years, p=0.05), more prior high-dose chemotherapies (p<0.001), while in the Campath group more bone marrow as stem cell source was used (p<0.001). No primary graft failure occurred in both groups. Patients receiving alemtuzumab had a significant faster engraftment of leukocyte (p=0.03) and of platelets (p=0.02) and a lower incidence of acute GvHD grade II-IV (24 vs 47%, p=0.05). However, after treatment with donor lymphocyte infusion due to persistent disease or mixed hematopoietic chimerism the difference of acute GvHD grade II-IV between alemtuzumab and ATG treated patients did not reach statistical significance (32 vs 47%, p=0.2). No difference in incidence of chronic GvHD was observed (25 vs 33%, p=0.6) More CMV seropositive patients in the alemtuzumab group experienced CMV reactivation (100% vs 47%, p=0.001). The cumulative incidence of treatment related mortality at 2 years for ATG and Campath was 29.3% (CI=17–50%) vs 28.5% (CI=15–54%), p=0.7. No significant difference could be observed in the estimated 2 years OS and PFS between ATG and Campath: 53% (CI:38–75) vs 45% (CI:28–73) and 29% (CI:16–54) and 36% (CI: 20–62), respectively. For PFS, in a multivariate analysis relapse to prior high-dose chemotherapy was the strongest negative factor: HR 2.9, p= 0.001. Including only those patients who did not experienced any relapse at time of allogeneic stem cell transplantation the Campath-group had a 2.5 fold higher risk of progression in comparison to the ATG group, but without reaching statistical significance (HR: 2.5, p=0.15). Ten out of 73 patients had KIR-ligand mismatch in GvH direction. While in patients without KIR-ligand mismatch the cumulative incidence of relapse at two years was 50%, none of the KIR-ligand mismatched patients relapsed so far (p=0.02). The immunosuppressive effect from Campath is stronger than ATG resulting in less acute GvHD, but requires more DLI procedures to control diseases and resulted in a trend to a lower PFS in chemosensitive patients. This preliminary data further implicated a major role of KIR-ligand mismatch transplantation in multiple myeloma
Pharmacogenetics of Cyclophosphamide in Patients with Beta Thalassemia Major Undergoing Bone Marrow Transplantation.
Authors: Poonkuzhali Balasubramanian , Salamun Desire , Shaji Ramachandran V. , George Biju , Mathews Vikram , Chandy Mammen , Krishnamoorthy Rajagopal , Srivastava Alok
Published: October 2019

Cyclophosphamide (Cy) is commonly used in conditioning therapy for bone marrow transplantation (BMT). The conversion of Cy to its first active metabolite 4-hydroxy cyclophosphamide (4-HCy) is mediated by several enzymes of the cytochrome P450 (CYP450) family [CYP2B6, 2C9, 2C19 and 3A4]. Since the pharmacokinetics (PK) of 4-HCy is formation rate limited, the AUC ratio of 4-HCy/Cy reflects better the degree of systemic exposure to 4-HCy. The PK of Cy and HCy exhibits up to 20 fold inter-individual variation in patients during conditioning. To understand the mechanisms underlying the variation in Cy metabolism, we analyzed the PK of Cy and 4-HCy in 40 consecutive patients with beta thalassemia major undergoing BMT and correlated them with the common polymorphisms of CYP450 2B6, 2C9 and 2C19 genes and the pre transplant hepatic status (Lucarelli class) of the patient. All patients received Cy at a dose of 50mg/kg as one-hour intravenous infusion for 4 days (day -5 to -2) after 4 days of busulfan (day-9 to -6). Levels of Cy and 4-HCy were measured using high performance liquid chromatography. Genotyping for CYP2B6 G516T, CYP2C9 *2, *3 and CYP2C19*2 and *3 were done using PCR-RFLP methods. There was a 4–20 fold inter-patient variation in the PK of Cy and 4-HCy. Mean AUC of Cy was 2288±1169 mg* h/ml, (range: 674–5126), while that of AUC of 4-HCy was 5.67±2.52 mg* h/ml (range: 0.817–11.17). Patients with wild type CYP2B6 G/G genotype had significantly higher Cy 4-hydroxylation than those with the homozygous mutant genotype (T/T).

CYP4502B6, 2C9, 2C19 genotypes and ratio of AUC 4HCy/AUC Cy

CYP2B6 G/G (n=15) G/T (n=8) T/T (n=16) t-test p value 0.0033±0.0014 0.003±0.0013 0.0023±0.0011 0.03 (G/G vs. T/T) CYP2C9 *1/*1 (n=25) *1/*2 (n=3) *1/*3 (n=9) *2/*3 (n=2) *3/*3 (n=1) 0.003±0.0016 0.0037±0.0009 0.0023±0.0011 0.0014, 0.0015 0.0073 0.076 (*1/*2 vs. *1/*3) CYP2C19 *1/*1 (n=26) *1/*2 (n=10) *2/*2 (n=3) 0.0026±0.001 0.0036±0.0012 0.0029±0.0013 0.016 (1/1 vs. 1/2)

One patient with homozygous mutant genotype CYP 2C9 3/3 showed 2.6 fold higher Cy 4-hydroxylation (0.0073 vs. 0.0026), while two patients with CYP2C9 2/3 genotype had 2 fold lower than the mean of the ratio for all 2C9 genotypes. Patients with CYP2C19 1/2 genotype showed significantly higher ratio than those with CYP2C19 1/1 genotype. No association was found between patients’ pre- transplant liver status (Lucarelli class) and Cy 4-hydroxylation. We conclude that 4-hydroxylation of Cy is significantly influenced by CYP2B6, 2C9 and 2C19 genotypes. This could explain the wide inter individual variation in Cy PK which should be correlated with toxicity and outcome of BMT.
Cytochrome C Related Caspase Activation Determines Treatment Response in Childhood B- Precursor ALL.
Authors: Meyer Lueder H. , Karawajew Leonid , Schrappe Martin , Ludwig Wolf D. , Debatin Klaus M. , Stahnke Karsten
Published: October 2019

Drug resistance and treatment failure in acute leukemia may be attributed to apoptosis resistance in leukemia cells since defects in apoptosis signal transduction are commonly acquired during malignant transformation. Expression analysis of single molecules with regard to clinical outcome could so far not identify common apoptosis defects with prognostic value in primary acute leukemias. We addressed the role of apoptosis signaling for the initial response to induction treatment in pediatric B precursor lymphoblastic leukemia by functional assays. Apoptosis signaling in response to survival factor withdrawal was assessed in 82 primary leukemia samples by measurement of the key apoptotic events, caspase-3 activation and mitochondrial cytochrome c release.

Caspase-3 activation directly correlated to the initial response to treatment assessed by the percentage of leukemia cells in bone marrow on day 15 (p = 0.015). Intact apoptosis signaling was indicated by a significant correlation of caspase activation and cytochrome c release was found especially in the groups of good responding patients (rs 0.375 – 0.502; p = 0.001). Interestingly, this correlation was completely absent in all groups of poor responding patients defined by insufficient leukemia cell reduction on day 8, 15 or 33.

The efficacy of apoptosis signal induction in the individual leukemia is expressed by the parameter CRAC (Cytochrome c Related Activation of Caspase-3), relating the extent of caspase activation to cytochrome c release in a single patient sample. Ten of twelve patients with prednisone poor response (chi square, p= 0.031) and all patients not achieving remission on day 33 (4 of 4) had negative CRAC values, indicating deficient cytochrome c related caspase activation. Furthermore, the CRAC negative group revealed significant higher percentages of leukemia cells in bone marrow on day 15 (n=37, mean 22.8) than the CRAC positive patients (n=30, mean 6.2, p = 0.004). In addition all four relapse patients showed negative CRAC values indicating a prognostic value beyond initial treatment response.

The data show that the intact relation of cytochrome c release and caspase activation indicating an intact apoptosome function is important for efficient remission induction treatment in childhood B precursor ALL. Assessment and quantification of this relation for individual patients by the parameter CRAC may serve as potential factor for treatment stratification.
Defective P38 MAPK Signalling Impairs Chemotactic but Not Proliferative Responses to SDF-1 in Acute Lymphoblastic Leukemia.
Authors: Bendall Linda , Baraz Rana , Juarez Julius , Shen Sylvie , Bradstock Ken
Published: October 2019

The chemokine SDF-1 regulates leukemic cell motility and proliferation but the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1-mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1, a subset of cases (18%) did not undergo chemotaxis in response to SDF-1 (unresponsive cases). These unresponsive cases also failed to increase b1 integrin-mediated adhesion to fibronectin or adhesion to bone marrow fibroblast layers. However the unresponsive cases could still elicit a calcium flux in response to SDF-1 and three of the four cases internalised the receptor, CXCR4, following exposure to SDF-1. In contrast, the CXCR4 antagonist, TC14012, inhibited proliferation of both responsive and unresponsive cases in stromal-dependent cultures, demonstrating that the unresponsive cases were still able to undergo proliferative responses to SDF-1. Examination of the signalling pathways activated by SDF-1 in responsive cells revealed increased phosphorylation of AKT, ERK and p38 MAPK 2 to 5 minutes following stimulation. However, cells from the unresponsive cases failed to phosphorylate p38 MAPK kinase when stimulated with SDF-1, while phosphorylation of AKT and ERK were comparable with that observed in responsive ALL cases. Inhibition of p38 MAPK by SB203500 completely inhibited the chemotaxis of responsive ALL cells to SDF-1 gradients suggesting that signalling through p38 MAPK is essential for ALL cell chemotaxis. Therefore it is likely that the absence of p38 MAPK phosphorylation in unresponsive cases underlies their lack of chemotaxis to this chemokine. The ability of the unresponsive cases to undergo SDF-1 driven proliferation in the absence of p38 MAPK phosphorylation suggests that, despite being absolutely required for chemotactic responses, induction of phosphorylation of p38 MAPK is not required for proliferative responses. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in NOD/SCID mice. This study suggests that signalling through p38 MAPK is required for ALL cell chemotaxis, but not for proliferation, and that chemotactic responses to SDF-1 are not essential for leukemic cell engraftment.
Efficient Thrombin Generation Requires Molecular Phosphatidylserine, Not a Membrane Surface.
Authors: Majumder Rinku , Weinreb Gabriel E. , Lentz Barry R.
Published: October 2019

Activation of prothrombin to thrombin is catalyzed by “prothrombinase” complex, traditionally viewed as factor Xa (FXa) in complex with factor Va (FVa) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through “channeling” (activation without release of an intermediate). We ask here whether the PS molecule itself, not the membrane surface, is sufficient to produce fully active human “prothrombinase” complex in solution. Both FXa and FVa bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FXa and FVa2 (light isoform of FVa), these proteins form a tight (Kd = 0.6 ±0.09 nM at 37°C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels show that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FXa and one FVa2, further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2x108 M− 1sec− 1) and via the same reaction pathway (50–60% channeling, rest via meizothrombin intermediate) as activation catalyzed by a complex assembled on PS-containing membranes (4.4x108 M− 1sec− 1). These results question the accepted role of PS-membranes as providing “dimensionality-reduction” and favor a regulatory role for platelet-membrane-exposed PS.
Outcome of Submyeloablative Allogeneic HSCT for Hematologic Malignancies: Development of a Prognostic Model Based on Pre-Transplant Characteristics.
Authors: Mehta Jayesh , Evens A. , Frankfurt O. , Gordon L. , Grinblatt D. , Kaminer L. , Meagher R. , Tallman M. , Williams S. , Winter J. , Singhal S.
Published: October 2019

Our aim was to identify pre-transplant variables affecting the outcome of submyeloablative HSCT in 63 patients (27–66 y, median 52) with NHL/Hodgkin/CLL (n=29), acute leukemia (n=19), and myeloma (n=15). Donors were HLA-matched sibs (n=37), 10/10 allele-matched unrelated (n=19), or 1-locus/allele mismatched (n=7). The conditioning was 100 mg/m2 melphalan with (n=42; no prior auto) or without (n=21; prior auto) 50 mg/kg cyclophosphamide. GVHD prophylaxis comprised cyclosporine-mycophenolate (HLA-matched sibs) or tacrolimus-mycophenolate (others). G/GM-CSF were not used. Supportive care was uniform. 16 patients died of transplant-related causes (TRM), and 35 relapsed (8 alive after salvage therapy). Of the variables analyzed in univariate fashion for effect on OS, the following were found to be significant and were entered into a Cox model for TRM, relapse, EFS and OS: age (83% <60 y vs 17% ≥60 y), performance status (33% 0 vs 41% 1 vs 25% ≥2), chemosensitivity (60% refractory vs 40% non-refractory), donor (63% sib vs 37% unrelated), HLA (89% match vs 11% mismatch), donor age (48% ≤45 y vs 52% >45 y), 108/kg CD3+ cells infused (49% <3 vs 51% ≥3), LDH (56% normal vs 44% increased), platelet count (40% <100 vs 60% ≥100), albumin (32% ≤3 vs 68% >3), and hemoglobin (24% <9 vs 76% ≥9). Relapse was higher with refractory disease (RR 3.2; P=0.003) and donor age >45 (RR 2.7; P=0.007), and TRM higher with PS ≥2 (RR 22.7 vs PS 0; P=0.001), donor age >45 (RR 5.3; P=0.013) and increased LDH (RR 8.6; P=0.003). The table shows factors influencing OS and EFS.

Variable OS DFS Adverse Favorable RR (95% CI) P RR (95% CI) P PS ≥2 PS 0 0.17 (0.06–0.47) 0.001 0.28 (0.11–0.78) 0.007 PS ≥2 PS 1 0.48 (0.24–0.98) 0.045 0.50 (0.24–1.03) 0.061 Increased LDH Normal LDH 0.29 (0.14–0.62) 0.001 0.43 (0.22–0.83) 0.012 Refractory disease Non-refractory disease 0.38 (0.15–0.92) 0.033 0.36 (0.17–0.77) 0.008 Donor age >45 Donor age ≤45 0.39 (0.19–0.77) 0.007 0.27 (0.14–0.53) <0.001

Scoring 1 point for each adverse factor (2 for PS ≥2), patients were divided into 4 groups: none (n=9), 1–2 (n=20), 3–4 (n=29), and 5 (n=5). The figures below show adjusted OS and EFS curves derived from the Cox analysis by the number of adverse variables. The figures below show actual OS and EFS Kaplan-Meier curves by the number of adverse variables.

Figure Figure

The figures below show actual OS and EFS Kaplan–Meier curves by the number of adverse variables.

Figure Figure

Interestingly, diagnosis, patient age and type of donor did not affect outcome. These data will allow better prediction of outcome in patients proposed to be treated with this regimen, and suggest that modification of therapy is needed for patients with ≥3 adverse variables to improve survival.
Tyrosine Phosphoproteomics of FLT3 Signaling.
Authors: Gu Tinglei , Nardone Julie , Lee Kim , Gygi Steven , Rush John , Comb Michael , Polakiewicz Roberto
Published: October 2019

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Recently, FLT3 has been implicated in the pathogenesis of infant and childhood ALL. FLT3 inhibition has been shown to kill ALL cells with high levels of FLT3 expression. Using immunoaffinity purification of phospho-tyrosine peptides followed by tandem mass spectrometry, we not only identified two new AML cell lines with activated FLT3, but also generated phosphotyrosine profiles for five cell lines that express either WT or ITD mutation of FLT3. We identified six novel tyrosine phosphorylation sites in FLT3. To further investigate the FLT3 pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in FLT3 inhibited versus uninhibited cell lines. Quantitative SILAC experiment identified over 100 proteins, whose tyrosine phosphorylation status are regulated by FLT3. Furthermore, it reveals common and distinct signaling pathways activated by either WT FLT3 in B-ALL or FLT3-ITD in AML. Our study represents a breakthrough in our understanding of FLT3 signaling network. It could have important implications for the design of novel therapeutic approaches and the identification of biomarkers for leukemia with activated FLT3.
Defective Engraftment of HSPC from C3aR−/ − Mice Reveals an Underappreciated Role of C3a-C3aR Axis in Stem Cell Homing to Bone Marrow.
Authors: Reca Ryan , Kucia Magda , Baran Jarek , Ratajczak Janina , Ratajczak Mariusz Z.
Published: October 2019

Recently we demonstrated that complement (C) is activated in bone marrow (BM) during conditioning for transplantation and hematopoietic stem/progenitor cells (HSPC) express on their surface the C3a receptor (C3aR) and in the presence of the third C component - C3 cleavage fragments (C3a and desArgC3a) respond more robustly to a chemotactic gradient of stromal-derived factor (SDF)-1 (Reca et al., Blood2003, 101, 3784). The molecular explanation for this phenomenon is a C3a mediated increase in the incorporation of CXCR4 into membrane lipid rafts what enables CXCR4 to interact better with small GTPases from the Rho/Rac family (Wysoczynski et al. Blood2005, 105, 40–48). To elucidate this phenomenon better and to learn more on the role of the C3a-C3aR axis in homing/engraftment of HSPC we studied i) engraftment of murine HSPC derived from C3aR-deficient mice into wild type littermates and ii) human HSPC on which C3aR was blocked by C3aR antagonist SB290157 into NOD/SCID mice. We noticed that wt mice transplanted with C3aR−/ − HSPC engrafted significantly worse compared to normal littermates. Accordingly, transplantation of the same numbers of Sca-1+ cells from C3aR−/ − mice into wt littermates as compared to transplantation of wt cells resulted in i) delay by ~5–7 days in recovery of platelets and leukocytes, ii) decrease in day 12 CFU-S, and iii) decrease in the number of CFU-GM progenitors detectable in the BM cavities at day 16 after transplantation. Similarly in parallel experiments, human CD34+ cells exposed to nontoxic doses of C3aR antagonist SB29007 engrafted worse in NOD/SCID mice (p<0.0001). Next, we studied the different steps of homing of HSPC and noticed that sensitization of cells to an SDF-1 chemotactic gradient was compensated in C3aR−/ − mice probably by the activation of another putative receptor for C3a, however, the C3aR was indispensable for optimal adhesion of HSPC to endothelium and SDF-1-dependent MMP-9 secretion. In conclusion, activation of the C cascade in BM during conditioning for transplantation exposes a natural neoantigen which is recognized by immunoglobulins activating C by the classical pathway. As a consequence, C3 cleavage product, C3a, activates the C3aR on transplanted HSPC increasing the SDF-1 mediated homing of these cells.
Sensitive and Quantitative Measurement of Gene Expression Directly from Peripheral Whole Blood, without RNA Isolation and Target Amplification.
Authors: Zheng Zhi , Luo Yuling , McMaster Gary
Published: October 2019

Peripheral blood gene expression analysis is increasingly used for diagnosis and prognosis of hematological diseases, as well as for surrogate biomarker discovery in a wide range of non-hematological disorders. However, a critical issue concerning the potential clinical application of these research findings relates to the validity and reproducibility of the blood mRNA quantitation results. Current gene expression technologies often depend on multiple steps: 1) blood isolation; 2) RNA purification and 3) subsequent enzymatic reactions, which affect the accuracy and consistency of results. We describe an assay to measure single- and multi-plexed gene expression directly from whole blood without RNA purification and target amplification. The hybridization-based assay uses branched DNA signal amplification technology with a whole blood lysis protocol that preserves the RNA integrity. The assay is sensitive enough to quantitatively measure genes expressed at low levels in a minority of cells from less than 30ul of whole blood. The coefficients of variations are less than 10% and the dynamic range is 3–4 logs for both singleplex and multiplex formats. The assay signals are several times higher than purified RNA from an equivalent amount of blood. Blood proteins, genomic DNA and reticulocyte mRNAs do not interfere with the assay. The assays are compatible with common anticoagulants and Paxgene treated samples. However, we found the Paxgene induced expression of antiapoptotic genes during processing of the whole blood. We used the multiplex assay to evaluate the impact of common blood processing on the expression of a panel of 30 cytokine and apoptosis genes known to be sensitive to ex vivo purtabation. Minimal impact was found with RBC lysis, followed by whole blood RNA extraction, PBMC isolation and Paxgene stabilization. The lowest correlation of expression was found between RNA extracted from whole blood and RNA extracted from same blood treated with Paxgene (R square=0.77). We believe this assay will contribute to fundamental and therapeutic applications where quantitative gene expression and/or throughput are required.
Preliminary Phase II Results of SGN-30 (Anti-CD30 Monoclonal Antibody) in Patients with Cutaneous Anaplastic Large Cell Lymphoma (ALCL).
Authors: Duvic Madeleine , Kunishige Joy , Kim Youn H. , Foreno-Torres Andres , Pinter-Brown Lauren C. , Rarick Mark U. , Zic John , Worobec Sophie M. , Foss Francine , Flessland Karen , Barton Jeremy
Published: October 2019

Background: SGN-30 is a chimeric monoclonal antibody (mAb) directed against the CD30 antigen which is expressed on Hodgkins and non Hodgkins Lymphoma. SGN-30 has been well tolerated in phase I and phase II studies and objective responses have been seen in systemic ALCL.

Methods: Adult CD30 positive cutaneous ALCL patients who have progressed after treatment with radiotherapy and/or systemic therapy, ECOG performance status 0–2, are treated in a multicenter, single arm phase II study. SGN-30 at 4 mg/kg is administered by IV infusion monthly for a maximum of 6 consecutive doses. Evaluation of response (Physician’s Global Assessment) is performed 3–4 weeks after each infusion. In the absence of an objective response after two doses, patients may receive SGN-30 at 12 mg/kg for the remaining infusions.

Results: Patient Characteristics: 5 patients (4 male, 1 female) with median age 63 years (range 42–79) have been enrolled. Median number of prior therapies is 3 (range 1–4). All patients had at least 1 prior systemic therapy, and 3 of 5 patients (60%) had received at least two of the following therapies: Targretin, CHOP, or radiotherapy. 4 patients received 6 doses of SGN-30; of these 1 patient increased to 12 mg/kg at dose 3, 1 at dose 5, and 2 at dose 2. One patient received a single 4 mg/kg dose. Efficacy: 4 of 5 patients (80%) achieved a response; 1 CR and 3 PR (2 designated as “Marked Improvement” and 1 as “Almost Clear”). The patient with a CR had a history of Mycosis Fungoides and developed CD30+ ALCL. He had complete resolution of trunk lesions after 5 doses of SGN-30, received one additional dose of SGN-30, and maintained a durable CR of > 40 days. Of the 3 patients with PR, 1 attained a response after one dose, and 2 after 3 doses. One patient with concurrent LyP received a single dose of SGN-30 9 days after discontinuing methotrexate, experienced a worsening of his disease, and came off the study with PD. Tolerability: The most common adverse events in the 5 patients included pedal edema, arthralgia, and insomnia, each of which occurred in 2 patients. Of the 6 grade 3/4 adverse events, 4 were considered to be unrelated to SGN-30, and the relationship of 2 were unknown (pruritis and increased number of lesions). There was 1 SAE, an 80 year old male hospitalized with pneumonia after 6 doses of SGN-30 died on study of myocardial infarction secondary to aortic stenosis, considered unrelated to study drug.

Conclusions: Preliminary data indicate the drug is well tolerated and shows antitumor activity. Recruitment into the study continues.
Type of BCR-ABL Transcript Predicts Response to Imatinib Mesylate.
Authors: Sharma Pratibha , Kumar Lalit , Kochupillai Vinod , Mohanty Sujata , Sharma Atul , Seth Tulika
Published: October 2019

Imatinib Mesylate, a molecular target therapy is the first line treatment for all CML patients. We prospectively studied 50 CML patients (37 pre-treated with interferon Alfa) for response to Imatinib. Patients median age was 32 years, range, 11 to 62 years with male to female ratio being 2.5: 1. All patients were in chronic phase and received 400 mg imatinib daily for a mean time period of 18 months (range 3 to 38 months). Patients were monitored closely for clinical, hematological, cytogenetic & molecular response. Bone marrow examination was done at 3, 6, 9 then 4–6 months interval for cytogenetic response (CGR) and also for presence of BCR-ABL transcript types (b2a2, b3a2, e1a2 and e19a2) by multiplex and nested RT-PCR technique. All patients achieved complete hematological remission with in 3 months of starting Imatinib. 20/50 patients achieved Major CGR (complete -8, Partial CGR-12). Of these, 12/20(60%) patients had b2a2 transcript, 3/20 (15%) b3a2 transcript and 5/20 (25%) had both the transcripts. 24/50 had minor CGR (b2a2-10/24 (41.6%), b3a2-14/24 (58.3%). Remaining 6 patients had no Cytogenetic response (4/6-b3a2, 2/6-b2a2). Patients with BCR-ABL transcript b2a2 had a significant probability of achieving Major cytogenetic response 12/50(24%) compared with b3a2 transcripts, 3/50 (6%), p<0.001. However, there was no significant correlation between transcript type and spleen size, Hb, WBC, platelets counts. These initial observations are very provocative and suggest that site of breakpoint in major breakpoint cluster region might be predictive of cytogenetic responses to Imatinib Mesylate. We are continuing to accrue more patients in this study to confirm these results.