bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab?.
Authors: Terui Yasuhito , Sakurai Takuma , Mishima Yuji , Mishima Yuko , Sugimura Natsuhiko , Kojima Kiyotsugu , Yokoyama Masahiro , Hatake Kiyohiko
Published: April 2020

Purpose: Although rituximab is commonly used as induction and maintenance therapy for CD20+ malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance.

Methods: To investigate whether CD20 mutations affect the response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, peripheral blood or bone marrow of 48 patients with NHL using magnetic activated cell sorting (MACS). CD19+/CD20+ cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The resulting PCR products were sequenced, subcloned into the mammalian expression vector pTARGET, transfected into K562 cells and CD20 expression was examined by flow cytometry and laser scanning confocal microscopy.

Results: In all 48 patients, overall response rate (CR+CRu+PR) to rituximab was 93.8% (45/48), but two cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in two CD20 domains - the third transmembrane domain and the C-terminal cytoplasmic domain. One patient had point mutations in the transmembrane domain, three cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. Thirty-eight patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain.

Conclusions: This study suggests that point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab.
BCR-ABL Is Not an Immunodominant Antigen in CML.
Authors: Grünebach Frank , Mirakaj Valbona , Müller Martin R. , Brümmendorf Tim , Brossart Peter
Published: April 2020

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder, which originates from pluripotent hematopoietic bone marrow progenitor cells. It is characterized by excessive proliferation of the granulopoiesis in the bone marrow. In approximately 90% of the patients with CML the Philadelphia chromosome (Ph) is the characteristic cytogenetic hallmark. The Ph is a shortened chromosome 22, which arises from a acquired reciprocal translocation between chromosome 9 and 22 (t(9;22) (q34;q11)). This translocation fuses the bcr gene with part of the c-abl gene, which encodes a protein-tyrosine kinase. The chimeric bcr-abl oncogene is translated into a 210-kDa chimeric protein (p210) which exhibits constitutive ABL kinase activity. In the present study, we analysed the involvement of the BCR-ABL protein in the induction of antigen-specific cytotoxic T lymphocytes (CTLs) in order to develop an immunotherapeutic approach targeting this neo-antigen in patients with CML. To accomplish this, we generated dendritic cells (DCs) in vitro and electroporated them with various sources of RNA harbouring the chimeric bcr-abl transcript. These genetically engineered DCs where used as antigen presenting cells (APCs) for the in vitro induction of CTLs.

By applying this approach we found that the CTLs induced by DCs transfected with RNA extracted from bcr-abl-positive K-562 cells or CML blasts do not recognize epitopes derived from the chimeric BCR-ABL fusion protein. In contrast, they were able to lyse autologous DCs electroporated with RNA isolated from patients with acute myeloid leukemia (AML), indicating that antigens shared among these malignant cells are involved and recognized by these CTLs. However, we successfully generated BCR-ABL-specific CTLs by DCs electroporated with in vitro transcribed bcr-abl-RNA. In patients with CML in complete cytogenetic remission during IFN-α treatment there was some reactivity against BCR-ABL in IFN-γ ELISPOT assays, which was weaker as compared to proteinase 3 (PR3)- or Prame-directed responses supporting the observations obtained in healthy donors.

In summary, BCR-ABL is processed and presented by Ph+ cells but is not the immunodominat antigen that induces CD8+ T lymphocytes in competition with other tumor-associated antigens. Nevertheless, APCs transfected with pure bcr-abl-IVT in vitro are capable of inducing BCR-ABL specific CTLs that efficiently lyse Ph+ target cells.
2005 Update on Accelerated FDA Approval of Drugs Used To Treat Hematologic Malignancies from the Research on Adverse Drug-Events and Reports (RADAR): Rate of Regular Approval Conversion Remains Low.
Authors: Bennett Charles L. , Zagory Jessica A. , McKoy June M. , McCaffrey Kathryn R. , Lyons E. Allison , Belknap Steven
Published: April 2020

Background: At ASH 2004 we reported that prior to 2004, eight drugs had received accelerated approval (AA) for ten hematologic oncology indications, only one had converted to full approval, and three serious adverse events associated with two of these drugs were identified post-approval. Following our presentation, Congressman Ed Markey (D-Massachussets) in June 2005 issued a report (“Conspiracy of Silence”) that raised similar concerns about the low rates of conversion from accelerated to regular approval and the failure of the FDA in ensuring that drug companies complete the required post-marketing studies. We update the experience with accelerated FDA approval for hematologic oncology.

Methods: Information was obtained from the FDA on new drug applications and supplements for new uses, package inserts, and medical literature reviews.

Results: Of 11 drug indications that received AA for hematologic oncology between 1995 and 2005, AA was generally granted for 9 based on clinical trials with <100 patients that identified high response rates. In the past year, none have applied for conversion to regular approval. Bortezomib received regular approval for an indication different than that granted for AA. Three serious adverse drug reactions were identified for two indications (gemtuzumab ozogamicin, imatinib for GIST) within 2 years of AA approval.

Conclusion: Since 2004, one new drug received AA for a hematologic oncology indication, one drug received regular approval for an indication different from that indicated under AA, and although 1 more year has elapsed, conversion has yet to occur for nine AA hematologic oncology indications. It is imperative that the FDA establish clear milestones regarding conversion to regular approval at the time AA is granted.

Drug Name (AA Date; Years Lapsed Since AA) AA Indication Years to AA/Regular Approval N in Trial (Overall Response Rate) *Regular approval granted for myeloma failing one treatment Denileukin difitox (2/99; 6.5) T-cell lymphoma 1.17/- 71 (30%) Cytarabine liposomal (4/99; 6.3) Lymphomatous meningitis 0.46/- 14 (71%) Gemtuzumab ozogamicin (5/00; 5.35) CD33 positive AML 0.58/- 142 (30%) Alemtuzumab (5/01; 4.3) CLL 1.375/- 93 (33%) Imatinib mesylate (5/01) CML 0.25/2 864 (80%) Ibritumomab tuixetan (2/02; 3.5) Low-grade NHL 1.3/- 157(68%) Imatinib mesylate (2/02; 3.5) GIST 0.29/- 147 (54%) Imatinib mesylate (12/02; 2.7) CML peds 0.5/- 39 (67%) Bortezomib (5/03) Multiple myeloma w/ 2 prior chemo treatments* 0.46/2 193 (35%) Tositumomab (6/03/2.2) NHL 4.0/- 59 (71%) Clofarabine (12/04; 0.7) Relapsed/refractory ALL peds 0.75/- 49 (20%)
Risk Factors of HLA-Haploidentical Transplantation Using the Combination of Unmodified Marrow and CD6-Depleted G-CSF Mobilized Blood Cells.
Authors: Kolb Hans-Jochem , Bigalke Iris , Tischer Johanna , Hill Wolfgang , Falk Christine , Simoes Belinda , Schmid Christoph , Ledderose Georg
Published: April 2020

Allogeneic stem cell transplantation from HLA-haploidentical family members has been studied in 80 patients for the treatment of hematological malignancies in advanced stages. The protocol involves unmodified marrow on day 0 and CD6-depleted G-CSF-mobilized blood cells on day 6. Engraftment was seen in patients treated with myeloablative (12 Gy) and non-myeloablative (4 Gy) doses of total body irradiation. Graft-versus-host disease (GVHD) was mild or absent in 53% and severe GVHD did not develop after changing to a more effective method of depletion of CD6-positive cells. Acute GVHD resolved more often and chronic GVHD was less severe than in HLA-identical sibling transplantation. Here we evaluated survival and relapse risk. Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) was the diagnosis in 41 patients, acute lymphoid leukemia (ALL) in 19 patients, chronic myelogenous leukemia (CML) in 2, chronic lymphocytic leukemia (CLL) in 3, high grade lymphoma (NHL) in 14 and severe aplastic anemia (SAA) in one patient. The patient’s age ranged from 17 to 62 years (median 36.5 yrs), for 65 patients a relative of first degree and for 15 patients a second degree relative was the donor. The major cause of death was recurrence of the disease (29 pts.), 7 patients died early and 22 pts. died of complications (infections 12, GVHD 3, pulmonary compl. 2, PTLD 3, cerebr. hemorrhage 1, liver tox. 1). Survival was significantly better in male patients with female donors (2 yr survival 36% vs 13%; p=0.001), patients transplanted with cells from their mother rather than from the father (2 yr survival 55% vs 8%; p=0.02) and patients transplanted in an earlier phase (2yr survival 50% vs 15%; p=0.048). Patients transplanted for ALL (2 yr. survival 31%) fared better than patients with AML/MDS (2yr survival 23%)and patients with NHL (2 yr. survival 12.5%) (p=0.08). Age, number and type of HLA-mismatches, as well as the depletion method had no effect on survival and on relapse risk. The favorable effect of the female gender of the donor on survival was still significant in multivariate analysis after adjustment for the stage of the disease and the diagnosis (p=0.01). The possibility of non-inherited maternal antigens (NIMA) inducing tolerance was tested in siblings sharing the paternal haplotype. There was a weak effect in the host-versus-graft direction (p=0.06), but no effect in the graft-versus-host direction. The risk of relapse was lowest in patients transplanted with stem cells from their mother as compared to those transplanted from their father (p=0.002). Female gender of the donor was favorable even in patients without GVHD. In summary female donors are preferable to male donors for control of leukemia and survival.
Bisphosphonate Related Osteonecrosis (BRON) of the Jaw: Single Institutional Update.
Authors: Mehrotra Bhoomi , Ruggiero Salvatore L.
Published: April 2020

BACKGROUND: Bisphosphonate therapy is indicated in the treatment of patients with symptomatic bony disease secondary to multiple myeloma, solid tumors and in the chronic management of osteoporosis. We have previously reported our early experience with bisphosphonate related osteonecrosis of the jaw (J Oral Maxillofac Surg2004; 62:527–534). This entity is now being recognized more commonly by treating hematologists/ oncologists and dental medicine community and the incidence of this complication has been reported between 4–10% among those patients receiving chronic intravenous bisphosphonate therapy. BRON is associated with significant morbidity in cancer patients and often requires symptomatic management for local palliation. The purpose of our current study is to update our previously reported experience at our institution and to review preventative and symptomatic management of this entity.

METHODS: We performed a retrospective analysis of patient records identified to have BRON at our institution. Institutional Review Board approval was obtained for this study. Information regarding age, gender, indication for bisphosphonate treatment, type of bisphosphonate agent used, clinical manifestation and management of osteonecrosis were recorded.

RESULTS: One hundred and two patients with BRON were identified at our institution. Median age was 67 years (range 40–89); Males: n=31; Females: n=71; Indications for bisphosphonate therapy: Multiple myeloma (n=40); Breast cancer (n=39), Prostate cancer (n=6), Uterine sarcoma (n=1), Connective tissue disorders (n=2), osteoporosis (n=14). Prior bisphosphosphonate therapy included: Pamidronate (n=28); Zoledronate (n=29); Pamidronate and Zoledronate therapy (n=31), and oral bisphosphonates (n=14). 24 of 102 patients had also received steroid therapy at some time during their clinical course. Most common presentation was a non healing extraction socket. Forty of 102 patients presented after surgical trauma to the jaw bone. Biopsies, when performed, confirmed the presence of osteonecrosis. No patients in this series had received radiation therapy to the jaw or had evidence of metastatic disease to the jaw bone. Surgical debridement was neccessary in 28 of 102 patients for local control of symptoms. Other patients were managed conservatively with antibiotics and local irrigation.

CONCLUSION: Long term intravenous and oral bisphosphonate therapy is associated with osteonecrosis of the jaw bone. Surgical trauma to the jaw bone may be associated with poor post-operative healing in these patients. Physicians and patients need to be aware of this significant potential complication of this therapy which may result in significant morbidity and impaired quality of life for these patients. Most patients can be managed conservatively, however, a significant fraction require surgical debridement for local palliation.
Boosts with Highly Purified Stem Cells Can Improve Hematopoietic Regeneration after Allogeneic Transplantation from Alternative Donors.
Authors: Lang Peter , Greil Johann , Koch Markus , Bader Peter , Pfeiffer Matthias , Berger Rebecca , Klingebiel Thomas , Wernet Dorothee , Niethammer Dietrich , Handgretinger Rupert
Published: April 2020

In allogeneic transplantation it is a major goal to maintain a stable long term engraftment. However, several factors like viral infections, drug toxicity or low stem cell numbers may hamper hematopoietic recovery or may even cause a late graft failure after successful initial engraftment. Hematopoietic growth factors or additional stem cell donations (boosts) may help to overcome this problem. In this retrospective analysis, the efficacy of boosts with highly purified peripheral stem cells either from closely matched unrelated or from haploidentical related donors was evaluated in pediatric patients with stagnant hematopoiesis (<1500 leucocytes or <50 000 platelets between 30–90 days posttransplant, n=11) or with initially increasing and then decreasing hematopoiesis (n=3). Patients with Graft rejection or hemophagocytosis were excluded. Diagnoses were: acute leukemias (n=10), nonmalignatn diseases (n=4). Most boosts were taken from the original graft: if the grafts contained >10x106 (unrelated donors) or >20x106/kg BW CD34+ cells, surplus stem cells were purified with an immunomagnetic selection method using antiCD34 or antiCD133 coated microbeads and cryopreserved. Three haploidentical donors were asked for a second donation. Our aim was to maintain stable and complete hematopoiesis after transplantation without inducing acute or chronic GvHD.

A total of 18 boosts were given between 30 and 290 days after transplantation with a median number of 5 (0.6–34) x 106 CD34+/CD133+ progenitor cells/kg and only 2200 (100–25000) residual T-cells/kg. All patients had a complete donor chimerism and no reconditioning or posttransplant pharmacological immunosuppression was administered. Patients who received G-CSF were excluded. Due to the low number of T cells, acute GvHD >grade I and also chronic GvHD could be completely avoided even in haploidentical donors.

Leukocyte counts, neutrophile counts, lymphocyte counts (and platelet counts in patients with thrombocytopenia) within the week before infusion of the boosts were compared with cell counts 4 and 8 weeks after infusion, using the paired T-test. A significant increase of all cell counts was observed (medina numbers pre boosting: 900/μl, 312/μl, 142/μl; 4 weeks post boosting: 2065/μl, 1264/μl, 317/μl; 8 weeks post boosting:2570/μl, 1780/μl, 385/μl; p<0.05 for all comparisons). Furthermore, independence from platelet transfusion could be achieved.

Conclusions: Boosts with purified stem cells and low T-cell contamination (<10000 T-cells/kg) can be administered after transplantation without the risk of inducing GvHD, even in mismatched donors. No pharmacological immunosuppression was necessary. Thus, the method represents a safe tool and can improve donor derived hematopoiesis.
Quality of Reporting of Harms in the NCI Sponsored Phase III Hematological Malignancies Trials.
Authors: Soares Heloisa P. , Kumar Ambuj , Djulbegovic Benjamin
Published: April 2020

Background: To make informed decisions, patients and physicians need accurate information both on treatment benefits and harms. However, reports of clinical trials typically focus on description of benefits only. Research supported by the NCI account almost for 100% of all publicly sponsored phase III trials in the US, and is widely considered as the most important for advancement of treatment against cancer. Therefore, it is important to assess the quality of reporting of treatment-related mortality (TRM) and morbidity (TRMorb) in this setting.

Methods: We evaluated all randomized controlled trials (RCTs) that have been conducted by six NCI sponsored cooperative oncology groups (COGs). We included all trials that were completed by 2002. Here we focus on hematological malignancy trials. Data related to TRM and TRMorb were extracted from publications that reported benefits (i.e. survival or event-free survival). Data were pooled using meta-analytic techniques to assess if the harms were worse in experimental vs. standard treatments.

Results: We evaluated,114 hematological-malignancy trials. Seventy-nine trials studied leukemias, 32 lymphomas and 3 either multiple myeloma or myelodysplastic syndrome. Quality of reporting for the most important methodological domains (such as randomization methodology, power calculation, drop-out rates, use of intention-to-treat analysis) was high. Data related to TRM were extractable from 41 trials (36%) only. Overall, TRM accounted for less than 5% of all deaths and was shown to be significantly higher in patients allocated to innovative treatments [Odds ratio 1.93: 99%CI (1.11–1.76)]. TRM was similar according to the type of hematological malignancy or COG. Treatment-related morbidity data were reported in different non-standardized formats; therefore, it was not possible to perform a meta-analysis to give an accurate assessment of TRMorb in experimental and conventional treatment arms.

Conclusion: Treatment-related harms have been inadequately reported even in the currently best available research. Given the fact, that the users of research evidence can only rely on the available published reports, the NCI should urgently improve the requirment for the standards of reporting of clinical trials it funds.
Long-Term Survival Is Comparable between Palifermin-Treated and Placebo-Treated Patients (Pts) with Hematologic Malignancies (HM) Undergoing High-Dose Chemotherapy and Total Body Irradiation Followed by Autologous Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Spielberger Ricardo , Emmanouilides Christos , Bensinger William , Rong Alan , Cesano Alessandra , Stiff Patrick
Published: April 2020

Oral mucositis (OM) is a severely debilitating side effect of chemoradiotherapy that often causes significant pain, diminished quality of life, and increased risk of infections. Palifermin decreases the incidence and duration of severe OM in HM pts receiving myelotoxic therapy and HSCT. Palifermin safety and efficacy have not been established in the non-HM setting. Since the rHuKGF receptor is not expressed by HM, palifermin is not expected to interfere with long-term disease outcomes in this pt population.

Aim: We assessed palifermin’s effects on the long-term disease outcomes (survival, disease progression, secondary malignancies) in pts with HM.

Methods: Long-term follow-up data were pooled from a 1998 phase 2, double-blind, placebo-controlled study (n=86) and a 2000 double-blind, placebo-controlled phase 3 study (n=212). The analysis included 152 pts treated with palifermin and 146 pts treated with placebo. Pts were assessed at 6-month intervals during the first year and annually thereafter until death or loss to follow-up. The Kaplan-Meier (K–M) method provided estimates of the safety endpoints. Data reported here are as of August 2004.

Results: There were 298 pts (152 palifermin: 146 placebo) monitored for long-term follow-up. The median follow-up period was 23.3 months for palifermin and 23.5 months for placebo. The overall survival and progression-free survival curves (p=0.474 and p=0.253, respectively) are similar between the palifermin and placebo groups. Secondary malignancies occurred in only 6 of 152 (3%) palifermin and 5 of 146 (4%) placebo pts. All secondary malignancies were myelodysplastic syndromes: 9 patients with diagnoses of Non-Hodgkin’s lymphoma and 2 patients of Hodgkin’s Disease. The number of deaths was similar between the groups (30% palifermin; 27% placebo); most deaths occurred within 12 months of randomization and were attributable to the underlying HM disease.

Conclusion: Use of palifermin for the prevention of severe OM has shown no negative impact on long-term disease outcomes, including survival, in the HSCT setting for patients with HM.
Graft Versus Host Disease-Like Syndrome after Autologous Stem Cell Transplantation in CLL Patients Treated with a TBI/Cy/Alemtuzumab (CAMPATH-1H) High-Dose Regimen.
Authors: Zenz Thorsten , Dreger Peter , Kröber Alexander , Ritgen Matthias , Bunjes Donald , Döhner Hartmut , Stilgenbauer Stephan
Published: April 2020

Introduction: A high incidence of unexplained skin rashes after an alemtuzumab containing high-dose regimen led us to retrospectively review CLL patients who underwent autografting within the CLL3C/CLL3 trials of the German CLL Study Group (GCLLSG) at a single center (University of Ulm).

Patients and Methods: CLL patients who received an autologous SCT after high-dose regimens with TBI (12Gy) / Cyclophosphamide (120mg/kg) without (CLL3: TBI/Cy) or with alemtuzumab (103mg i.v.) (CLL3C: TBI/Cy/A) between 2000 and 2004. Before SCT the patients received cytoreduction with FC and PBSC graft mobilization with Dexa-BEAM + G-CSF.

Results: 27 CLL patients received an autologous SCT after high-dose regimens with TBI/CY (n=11, age 34–61) or TBI/Cy/A (n=16, age 34–65). A mean of 6.0 (±2.8) (TBI/Cy) and 7.6 (±6.1) (TBI/Cy/A) x10-6 CD34+ cells/kg were transfused. At a median follow up of 24 months, overall survival was not different in the two groups (TBI/Cy: 91%; TBI/Cy/A: 81%). Twelve of 16 (75%) patients (TBI/Cy/A) developed a skin rash 43–601 days after transplantation (not including immediate alemtuzumab side effects) and 7 (44%) were classified as acute GvHD (skin grade 1–3). Concurrent symptoms included conjunctivitis (n=4), sicca syndrome(n=5), and cholestasis (n=4). The incidence of GvHD among patients alive at 1 year was 58% (TBI/Cy/A) vs. 0% (TBI/Cy) (log rank p=0.0048). On skin biopsy, the findings were compatible with GvHD grade 1–2 in 5/5 patients. All patients with GvHD were treated with steroids and in two patients GvHD was severe enough to lead to further immunosuppression with cyclosporine, MMF or FK506. In 3 patients signs and symptoms resolved and 4 patients improved but continued to have mild skin rashes and symptoms. Immune reconstitution showed that the CD4/CD8 ratio was more than 12 times higher in the TBI/Cy/A group (8.0 vs 0.6). When comparing the patients with GvHD with patients not developing a skin rash in the TBI/Cy/A group, the difference was statistically significant (ratio 20.3 (95%CI 4.8–35.7) vs 1 (95%CI0.7–1.3), Mann-Whitney: p=0.00001). The high ratio was due to an extreme depletion of CD8 positive T cells.

Conclusion: The incorporation of alemtuzumab into the high-dose regimen prior to autologous SCT in CLL led to a high incidence of a GvHD-like syndrome primarily of the skin. The remarkable incidence of GvHD went along with the severe depletion of CD8+ T-cells leading to an abnormally high CD4/CD8 ratio. The further investigation of the underlying mechanisms may lead to a better understanding of the development of GvHD and autoimmune disease and serve as a model system of these disorders.
Non-Myeloablative Autologous Hematopoietic Stem Cell Transplantation (HSCT) for Relapsing-Remitting Multiple Sclerosis.
Authors: Burt Richard K. , Oyama Yu , Statkute Laisvyde , Quigley Kathleen , Weppner Catherine , Verda Larissa
Published: April 2020

We have previously reported that autologous HSCT using a cancer-specific myeloablative regimen (cyclophosphamide and total body irradiation) did not improve the neurologic disability (EDSS) score or even forestall progressive neurologic decline in patients with secondary progressive (axonal degenerative) multiple sclerosis. We, therefore, redesigned our approach to use a non-myeloablative regimen composed of agents specifically used to treat multiple sclerosis (cyclophosphamide and CAMPATH-1H) with infusion of cyclophosphamide / G-CSF mobilized autologous peripheral blood stem cells (PBSC) in patients with relapsing-remitting (inflammatory) multiple sclerosis. This regimen was designed for lymphoablation without myeloablation. Nineteen patients were treated. The regimen was well tolerated. No patient had either an early or late infectious events, 50% of patients never had a neutropenic fever. Of those who developed fever during transplant, fever lasted less than 24 hours and was related to CAMPATH-1H or stem cell infusion. One-half of the patients never required a RBC transfusion and one quarter never required a platelet transfusion. The mean day of WBC engraftment was day +8. The mean day of hospital discharge was day +10. CD4 recovery occurred by 6 months. The EDSS neurologic rating scale that varies from 0 (normal) to 10 (dead from neurologic disease) is evaluated every 6 months for 1 year then yearly. In fifteen patients with 6 or more months of follow-up, the EDSS has improved by at least one point in the majority (8 patients), remained unchanged in six and deteriorated in only one. A randomized multicenter trial, the Multiple Sclerosis International Stem Cell Transplant (MIST) trial comparing this approach to continued standard therapy for relapsing-remitting MS in patients with an enrollment EDSS of 2.0 to 6.0 is currently registering patients.
Dose Intensive Induction Chemotherapy Does Not Decrease Tumor Contamination of Autograft or Improve Survival for Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplant.
Authors: Sayani Farzana A. , Bahlis Nizar J. , Faris Peter , Savoie Mary Lynn , Chaudhry Ahsan , Jeje Oluyemi , Brown Christopher B. , Russell James A. , Stewart Douglas A.
Published: April 2020

Multiple dose intensive chemotherapy regimens have been used for induction/mobilization of stem cells for autologous stem cell transplantation (ASCT) in multiple myeloma. However, the exact role and regimens of such dose intensive chemotherapy has not been clearly defined. We therefore did a retrospective study to determine if dose intensive cyclophosphamide (Cyclo) 5.25 g/m2, etoposide 1.05 g/m2 and cisplatin 105 mg/m2 (DICEP) results in improved relapse rate and survival compared to standard mobilization with only Cyclo 2 g/m2. Between January 1998 and March 2004, a consecutive series of 57 newly diagnosed multiple myeloma patients receiving DICEP (38) or Cyclo (19) for in-vivo purging/mobilization were analyzed. Both groups were similar in regards to age and sex. There were no significant differences in IPI score, Durie-Salmon stage, B2 microglobulin, calcium, creatinine and albumin levels between treatment groups. Outcomes included time to relapse and time to death. Median follow up time was 799 days. Kaplan-Meier plots for time to relapse showed no significant difference (p=0.0992). Median relapse time in the DICEP group was 905 days (95% CI 580–1604) compared to 1112 days (95% CI 742-infinity) in the Cyclo group. Kaplan-Meier plots for overall survival showed no significant difference between both groups (p=0.8664). The median survival times have not yet been reached in either group and are not reported. Analysis revealed no discernable confounding risk factors. Effects of treatment on outcomes were not altered after adjusting for IPI score and Durie-Salmon staging using the stratified log-rank test. Small sample size and short duration of followup are potential limiting factors for this study, however, preliminary analysis of a larger sample of 91 multiple myeloma patients receiving either DICEP or a less intense induction/mobilization regimen, also revealed no significant difference in disease free or overall survival. Monoclonal plasma cell contamination of stem cell products was not significantly different between both groups (DICEP 42.9%, Cyclo 56.3%, p=0.5573). This study therefore suggests that the very intense DICEP induction/mobilization regimen results in no significant difference in survival outcomes. The more intense regimen does not significantly decrease tumor contamination of autograft stem cells. Overall, our experience suggests that novel induction therapies such as bortezomib, lenolidomide etc. should be pursued in preference to any further study of high dose cytotoxic chemotherapy induction.

Figure Figure
Tymic Output after Autologous Bone Marrow Transplantation (ASCT) HIV-Related Lymphoma.
Authors: Simonelli Cecilia , Spina Michele , Pratesi Chiara , Tedeschi Rosamaria , Rupolo Maurizio , Bortolin Maria Teresa , Zanussi Stefania , Michieli Mariagrazia , De Paoli Paolo , Tirelli Umberto
Published: April 2020

Background: One of the major concern for ASCT in HIV-positive patients (pts) is that post-transplant immunosuppression might worsen the immunosuppression and enhance the HIV replication. Therefore we analyzed whether the immune system of HIV-positive pts might support an immune recovery similar to the HIV-negative pts who underwent the same ASCT program.

Methods: The kinetics and the extent of immunereconstitution were assessed by measuring: TCR excision circles (TRECs), CD4, CD8, CD56, CD4, CD45RA, CD19 cells, and HIV pro and viraemia

Results: 15 HIV-positive and 7 HIV-negative pts with relapsed DBCL underwent ASCT. Before the induction therapy TRECs/106 PBMC mean value was 962±2183 and 2948±5485 in HIV-positive and HIV-negative pts respectively. The nadir was reached before ASCT (150±11 vs 927±1842). At one year the mean value of TRECs/106 PBMC returned to the baseline in HIV-negative pts, while in HIV-positive pts largely overcame the baseline (2440±2799). Before the induction therapy, HIV-positive pts showed a significant lower CD4 (174±110 vs 386±200) CD56 (86±129 vs 144±92) and CD4/CD8 ratio in comparison with HIV-negative pts. CD4 count nadir was reached during aplastic period in both groups. No differences were present in the dynamics of CD4, CD45RA, CD8 and CD56 recovery between the two groups. At one year CD4 count returned to the baseline value in HIV-negative pts while in HIV-positive pts overcame 70% the baseline value. Before ASCT, all HIV-positive pts were on HAART and HIV-viraemia was <50cp/ml in 10/15; 6 pts discontinued HAART, but HIV viraemia and proviraemia did not increase significantly during the overall observational period.

Conclusions: The dynamics of the immunereconstitution was similar in HIV-positive and HIV-negative pts, but the tymic output unexpectedly seemed to be enhanced in HIV-positive pts. Supported by ISS grants.
B-Cell Scaffold Protein with Ankyrin Repeats (BANK1) Is Differentially Expressed in ZAP-70 Negative Compared to ZAP-70 Positive Chronic Lymphocytic Leukemia Cells.
Authors: Dicker Frank , Schnittger Susanne , Schoch Claudia , Kohlmann Alexander , Liu Wei-Min , Kern Wolfgang , Prof Torsten Haferlach
Published: April 2020

The lack of somatic mutations of the immunoglobulin variable heavy chain (IgVH) gene has been established as poor prognostic marker for chronic lymphocytic leukemia (CLL) patients at early stage disease. Expression of the non receptor tyrosine kinase zeta chain associated protein (ZAP-70) was proposed as a surrogate marker for an unmutated IgVH, however, up to 30% discordant samples have been reported depending on the respective study. B cell receptor (BCR) mediated signaling is enhanced by ZAP-70 expression in CLL cells in vitro and ZAP-70 expression also tends to decrease the time from diagnosis to treatment irrespective of the IgVH status. Therefore, we wanted to identify differentially expressed genes between the ZAP-70 positive and negative CLLs by gene expression profiling of peripheral blood mononuclear cells (PBMCs) using Affymetrix microarrays (HG-U133 Plus 2.0). ZAP-70 expression was analyzed by quantitative real time PCR of CD19 purified (purity > 99%) PBMCs (n=62) using a LightCycler instrument. Expression of ZAP-70 mRNA was normalized against the housekeeping gene ABL and a relative quantitation against Jurkat T cells as a calibrator was performed. Results are expressed as normalized ratio and a cut-off of 0.5 normalized ratio gave the best correlation to the IgVH status with 77% concordant samples between ZAP-70 expression and the IgVH status. The discordant samples consisted of 5 unmutated IgVHs in the ZAP-70 negative group and 9 mutated in the ZAP-70 positive group. In a second step PBMCs of the same samples were analyzed by gene expression profiling and differentially expressed genes were identified by t-test. Among the two best genes that could be used in a classification algorithm (SVM) to distinguish between the 2 subsets with 92% accuracy were ZAP-70 and B cell scaffold protein with ankyrin repeats (BANK1). The expression of BANK1 was increased 3–4-fold in the ZAP-70 negative compared to the ZAP-70 positive CLL subset (P = 0,001). In the literature, BANK1 has been identified in human BCR expressing B cells and seems to be B cell restricted. In B cells the scaffolding protein BANK1 enhances BCR-mediated Ca2+-signaling, a signaling pathway that is also enhanced by ZAP-70 expression in CLL B cells. Based on these data we show that increased BANK1 expression correlates with a ZAP-70 negative status in CLL B cells. The functional consequences of BANK1 expression in the ZAP-70 negative subset of CLL B cells, which are usually associated with a more favorable prognosis, still need to be established further.
Superior Acute Myeloid Leukemia-Specific T Cell Responses Using Dendritic Cells Pulsed with Apoptotic Bodies, vs.Tumor Lysates or mRNA.
Authors: Xing Dongxia , Decker William K. , Li Sufang , Champlin Richard E. , MacMannis John D. , Komanduri Krishna V. , de Lima Marcos , Shpall Elizabeth J.
Published: April 2020

Dendritic cells (DCs), which hold promise for cancer immunotherapy have two distinct functions. Immature DCs internalize antigens; while mature DCs trigger native T cell activation. To date, no consensus has been reached concerning the optimal source of tumor antigen nor the mode of antigen uptake for efficient cross-priming of tumor specific response. Reports have demonstrated that human DCs can acquire relevant antigens and stimulate MHC class I-restricted cytotoxic T lymphocytes (CTLs) by phagocytosing apoptotic tumor cells. Apoptosis was established to be the critical trigger of cross-priming in this process. Apoptotic bodies form when cells undergo programmed cell death. In this study, three acute myelogenous leukemia in (AML) antigen preparations were compared in an autologous setting.

Apoptotic bodies, Tumor lysate Tumor mRNA

Methods: DCs were generated by CD14 magnetic selection of peripheral blood progenitor cells (PBPCs) and six days of culture in GM-CSF and IL-4. Tumor lysate was made by repeated freezing and thawing of patient AML cells (leukodepletion product >95% blasts); mRNA was extracted using a kit (Qiagen); apoptotic bodies were generated with UVB irradiation of a fraction of the same AML product. The rate of apoptosis was detected using Annexin V FITC flow cytometry. The DCs were loaded with mRNA by electroporation. The DCs were loaded with lysate and apoptotic bodies by incubation. Autologous T cells stimulated with DCs loaded with the three differing antigen preparations were compared for interferon(INF)-g production in ELISPOT and proliferation assays.

Results: The DCs pulsed with apoptotic bodies; lysate and mRNA expressed similar levels of the costimulatory molecules CD80, CD86, and theDC maturation marker CD83. The internalization rate of apoptotic bodies was about 20% detected by DAPI (label the apoptotic bodies) and DC83 double staining. The lysate pulsed DC and mRNA pulsed DCs were above 90% detected by flow of trace FITC- albumin and tracer GFP mRNA from previous experiments. Yet IFN-g secretion of T lymphocytes was statistically significant higher (P<0.05), using apoptotic body-loaded DC as compared to total lysate or mRNA. The T cell proliferation stimulated by apoptotic body-loaded DC was also better than that of lysate or mRNA loaded DCs (p<0.05).

Conclusions: These preliminary data demonstrate that apoptotic body-loaded DCs induced better Th1 T cell responses than DCs loaded with AML lysate or mRNA. One potential advantage conferred by the use of apoptotic body loaded DCs is that presentation of intracellular proteins in tumor lysates and mRNA that are irrelevant to the induction of anti-tumor responses are eliminated, which may lead to better antigen presentation and consequently an improved Th1 response.
Telomere Length by Tel-PCR in B Cell Malignancies.
Authors: Parker Anton E. , Snell Kimberley , Davis Zadie , Oscier David Graham
Published: April 2020

Previous studies have shown heterogeneity of telomere length among chronic B cell malignancies and within CLL. Longer telomeres are a feature of germinal center-derived tumours and mean telomere length is shorter in unmutated than mutated CLL. The relative contributions of telomere length pre-transformation, germinal centre experience and tumour cell proliferation in determining telomere length remains uncertain. We have used a recently introduced real-time quantitative PCR method to measure the factor by which the ratio of telomere repeat copy number to single gene copy number differs between a sample and that of a reference DNA sample. We compared relative telomere length in 4 types of B cell tumour whose VH genes may be either unmutated or mutated.

In 24 cases of mutated CLL mean relative length was 4.24 (range 0.16–24.13) compared to 0.81 (range 0.1–3.1) in 21 cases of unmutated CLL, p= 0.004. However, analysis of 8 mutated and 7 unmutated CLL cases using the poor prognosis Vh3-21 gene showed no significant difference between the two subsets. In non-CLL cases, the mean relative telomere length was 0.7 in 11 cases of B-PLL, 1.9 in 10 cases of MCL and 4.06 (range 0.2–22.7) in 29 cases of SMZL. In the latter 3 disorders there was again no significant difference in mean relative telomere length between mutated and unmutated cases. However, when SMZL patients were divided into 2 groups with relative telomere lengths above or below the median, 11/14 patients with stable disease had long telomeres whereas 10/15 patients with progressive disease had short telomeres, p= 0.025. We then compared 26 cases of stage A, mutated CLL, of whom 13 had stable disease for >10 years and 13 had progressive disease. There was no significant difference in telomere length at presentation between the 2 groups nor on follow up testing of samples from 7 patients from each group with a mean of 92 months between test samples. In contrast, 5 cases with stable, stage A, unmutated CLL for >8years had a longer mean relative telomere length of 2.15 (range 1.29–4.12) than the cohort of 21 unmutated cases with progressive disease described above p=0.002.

These data confirm the differences in mean telomere length among different B cell tumours and between mutated and unmutated CLL, but suggest that mutational status per se does not influence telomere length, while in SMZL and unmutated CLL, rate of cell proliferation is reflected in telomere length.
Enhanced Potency of Glycoengineered Anti-CD52 Monoclonal Antibodies (MAbs).
Authors: Dyer Martin J.S. , Moser Samuel , Brünker Peter , Bird Pru , Almond Neil , Püentener Ursula , Wheat Luise M.W. , Bolam Emma , Berrie Eleanor , Grau Roger , Buckby Emma , Kennedy Ben , Stebbings Richard , Hale Geoff , Umana Pablo
Published: April 2020

The CD52 MAb alemtuzumab (Campath) has been licensed for Fludarabine-refractory CLL. Like most commercial therapeutic MAbs, it is produced in mammalian cells (Chinese Hamster Ovary cells) and contains fucosylated carbohydrates attached to the Fc region. In contrast, a fraction of natural human serum IgG lacks fucose on their Fc-oligosaccharides and such antibodies bind with higher affinity to the FcgRIII receptors of immune effector cells. We have produced glycoengineered versions of alemtuzumab with high levels of non-fucosylated Fc oligosaccharides, increased binding affinity to FcgRIII and increased target-cell killing potency via ADCC. Glycoengineering was achieved by stable over-expression of genes encoding carbohydrate-modifying enzymes in an industrial-grade CHO cell line. Two glycovariants were produced: CAMGlyco1, enriched in non-fucosylated, hybrid-type Fc carbohydrates and CAMGlyco2, carrying non-fucosylated oligosaccharides both of hybrid- and complex-type (in approximately equal proportions). Both glycovariants had a proportion of non-fucosylated oligosaccharides leading to markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) in comparison with regular alemtuzumab. CAMGlyco1 mediated lower complement-dependent cytotoxicity (CDC) than CAMGlyco2. The activity of the two glycoforms was assessed in cynomolgus monkeys. The pharmacokinetics of both glycoforms were not significantly different from wild-type alemtuzumab. There was no increased cytokine release with either of the glycoforms. Both CAMGlyco1 and CAMGlyco2 appeared to deplete lymphocytes from both blood and lymph node better than wild-type alemtuzumab. Mean lymphocyte loss from peripheral blood following low dose treatment was 33.7%, 55.9% and 55.0% for alemtuzumab, CAMGlycol1 and CAMGlyco2, respectively (p<0.003). Mean lymphocyte counts per mm3 of lymph node biopsy were 4.6x105, 3.2x105 and 3.6x105 for alemtuzumab, CAMGlycol1 and CAMGlyco2, respectively. A Phase I/II clinical trial comparing CAMGlyco1 directly to alemtuzumab in patients with relapsed CLL has been initiated. Patients receive alternating doses of wild-type alemtuzumab and CAMGlyco1 and rates of decrease in peripheral blood lymphocyte count are monitored. Preliminary results show no increased toxicity. Glycoengineering of CD52 MAbs may allow for enhanced cytolytic efficacy in tumor therapy.
Increasing the Dose Intensity of Alemtuzumab (CAMPATH-1H) in Chronic Lymphocytic Leukemia (CLL): The CAM1515 Study.
Authors: Dyer Martin J.S. , Buckby Emma , Fermahan Pam , Walewska Renata , Kennedy Ben
Published: April 2020

Background: The doses and timing necessary for effective cytoreduction with monoclonal antibodies remain largely unknown. The human IgG1 CD52 MAb alemtuzumab is normally given intravenously (IV) three times per week at a standard dose of 30mg, following dose escalation. However, comparable results may be observed with fewer side effects using subcutaneously (SC) administered MAb, although the pharmacokinetics of SC administered MAb are slower than IV and therapy may have to be administered on a thrice weekly schedule for up to 18 weeks to achieve maximal efficacy. Efficacy with alemtuzumab has been shown to relate directly to serum levels of the MAb. We wished to assess whether more rapid cytoreduction with acceptable toxicities might be attained with daily alemtuzumab dosing without dose escalation.

Patients and methods: Patients with CLL (either as first-line therapy or at first or subsequent relapse) received SC alemtuzumab for 15 consecutive days at a dose of 30mg daily and thereafter three times per week for up to 8 weeks. To minimize adverse skin and possible systemic reactions, dexamethasone (12mg) was given along with acetaminophen (1g) for the first two injections only.

Results: 7 patients (aged 55– 73, 6 male 1 female) have been treated comprising 3 patients with Binet stage A progressive and 4 stage C. Use of steroid premedication for the first two doses virtually abolished all acute toxicities with only one patient showing any significant cutaneous reaction. CMV PCR was performed weekly. 4 patients showed low-level CMV (up to 7000 viral copies/ml) reactivation in the absence of any symptoms. All were treated successfully with oral valganciclovir 900mg bd whilst continuing with alemtuzumab without interruption. All 7 patients showed rapid reductions of peripheral blood lymphocytes as shown in the figure. Rates of drop in lymphocye count were much more rapid with daily than with conventional thrice weekly dosing schedules. Despite the more rapid reductions in lymphocytes, no patient developed tumor lysis. All patients showed significant reduction in bone marrow infiltration after the 15 days of daily therapy although a further 8 weeks of therapy was necessary to induce CR.

Conclusions; these data indicate that daily SC dosing with alemtuzumab results in rapid cytoreduction without increased adverse events. Prolonged daily dosing may result in enhanced efficacy of alemtuzumab in CLL.

Figure Figure
Pharmacokinetic Analysis of Subcutaneous Alemtuzumab as Consolidation Therapy To Achieve MRD-Negative Responses.
Authors: Regazzi Mario , Montagna Michela , Avanzini Antonia , Scarpati Barbara , Colosimo Anna , Veronese Silvio , Tedeschi Alessandra , Miqueleiz Sara , Ricci Francesca , Intropido Liliana , Morra Enrica , Montillo Marco
Published: April 2020

Persistence of minimal residual disease (MRD) after therapy has been shown to be a powerful prognostic factor for relapse among patients with chronic lymphocytic leukemia (CLL). Treatment with the anti-CD52 monoclonal antibody alemtuzumab (Campath®), either as a single agent or in combination with fludarabine, has been shown to induce MRD-negative responses, even among patients who are not responsive to traditional therapeutic interventions. Dramatically improved responses and MRD negativity have also been achieved using alemtuzumab as consolidation therapy, to purge MRD after initial therapy with a fludarabine-based regimen. The pharmacokinetics (PK) of alemtuzumab administered subcutaneously to achieve MRD negativity after induction therapy with fludarabine are reported here. Of 41 patients (median age, 54 years) who received consolidation therapy with alemtuzumab, all 16 patients participating in the PK analysis were MRD-positive after receiving induction therapy as measured by PCR consensus primer. At least 8 weeks following the completion of induction therapy, dose escalated alemtuzumab 10 mg was administered for 6 weeks, 3 times a week. Plasma samples were collected from 16 patients on Days 1, 3, 5, 15, 17, 22 and 31, and ELISA was used to assess the PK of alemtuzumab. On Day 15, blood samples were drawn at 1, 2, 3, 4, 6, 8 and 12 hours after alemtuzumab administration. Alemtuzumab plasma concentrations gradually increased during the first 2 weeks, and reached concentrations 25.5-fold higher on Week 3. The median pre-dose concentrations (Cpre-dose) were: 0.01 μg/mL (range, 0–0.07 μg/mL) on Day 3, 0.58 μg/mL (range, 0–1.2 μg/mL) on Day 15, 0.51 μg/mL (range, 0–2.61 μg/mL) on Day 17, and 1.09 μg/mL (range, 0.19–3.08 μg/mL) on Day 31. The median Cmax plasma concentration was 1.05 μg/mL (range, 0.097–1.75 μg/mL) and the median Cpre-dose was 0.58 μg/mL (range, 0–1.2 μg/mL). An analysis was performed to correlate quantitative response as represented by polyclonal complete remission and AUC0–12 values. Preliminary data indicate that the systemic exposure associated with effective therapy corresponds with an AUC0–12 value higher than 5 μg•h/mL on Day 15 of the administration schedule. Therefore, complete response (CR) rates, expressed as percentage of patients with AUC0–12 values correlating with effective treatment, increased with higher levels of systemic exposure to alemtuzumab.