ALBERT

Description: A Rhesus D (RhD) red blood cell phenotype with a weak expression of the D antigen occurs in 0.2% to 1% of whites and is called weak D, formerly Du. Red blood cells of weak D phenotype have a much reduced number of presumably complete D antigens that were repeatedly reported to carry the amino acid sequence of the regular RhD protein. The molecular cause of weak D was unknown. To evaluate the molecular cause of weak D, we devised a method to sequence all 10RHD exons. Among weak D samples, we found a total of 16 different molecular weak D types plus two alleles characteristic of partial D. The amino acid substitutions of weak D types were located in intracellular and transmembraneous protein segments and clustered in four regions of the protein (amino acid positions 2 to 13, around 149, 179 to 225, and 267 to 397). Based on sequencing, polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction using sequence-specific priming, none of 161 weak D samples investigated showed a normal RHD exon sequence. We concluded, that in contrast to the current published dogma most, if not all, weak D phenotypes carry altered RhD proteins, suggesting a causal relationship. Our results showed means to specifically detect and to classify weak D. The genotyping of weak D may guide Rhesus negative transfusion policy for such molecular weak D types that were prone to develop anti-D.

Description: Abstract 292 Introduction: CLL cells derive essential cues from their microenvironment, that may be targets for therapy. To this end the immunomodulatory drug Lenalidomide has shown remarkable clinical activity in monotherapy trials in CLL. However, tumor lysis and tumor flare have been major obstacles in development and marked and unexplained differences in the individual tolerance of the substance remains an unsolved problem. Furthermore, the potential for interaction with standard treatment for CLL is unknown. We employed the combination of Fludarabine and Rituximab for early reduction of tumorload and used it as a backbone to establish a tolerable Lenalidomide dose in combination. Study design: In the induction phase a maximal tolerated dose (MTD) of Lenalidomide in combination with FR was to be determined during 6 cycles of Fludarabine (40mg/m2 po d1-3 q28d) and Rituximab (375mg/m2 iv d4 cycle 1; 500mg/m2 iv d1 cycles 2–6, q28d). In cycle 1 Lenalidomide was added day 7–21 at 2,5 mg. Toxicity permitting, Lenalidomide dose was escalated to 5, 10, 15, 20 and 25mg d1-21 over cycles 2–6. Data from this phase are presented in this planned analysis. Data from a 6 month Lenalidomide/Rituximab maintenance phase will be presented later. Results: The median age of the 45 recruited patients was 66 years (range 43–79). Half of the patients were in stages Rai III/IV and the median β2-MG was 4.4 mg/l. At least one high risk feature from CD38, FISH analysis and mutation status was present in 64% of patients. Five patients stopped treatment during induction (Two due to rashes, two as patient's choice and one due to early Richter's transformation). No systematic toxicity determining an MTD, the primary study endpoint, was found. In striking contrast to a small previous report, 34% of our patients proceeded through dose escalation steps as planned to receive a dose of 25mg of Lenalidomide with their last cycle of FR. The individual MTD was equal or higher than 10mg in 73% of ITT patients and 71% in this group were dose-limited due to individual differences in myelotoxicity. In ITT analysis 27% of patients had an MTD of less than 10mg. Grade 3 and 4 neutropenia was expected in this combination and observed in 88% of patients in any cycle. While it was not used as a dose limiting toxicity per se, 42% of patients were dose-limited due to myelotoxicity at some level. Infectious episodes of grade 3 severity were observed in 5 patients (11%), resulting in a relatively mild rate given the observed myelotoxicity and the phase I/II design. Surprisingly, 1/3 of the patients experienced greater than G2 skin toxicity and this was deemed dose-limiting in nine patients. No tumor lysis or greater than G2 flare reactions were observed. Full response assessment for induction treatment is available for 39 patients. Complete responses were observed in 49% and partial responses in 38% of the ITT population. In 35 patients flow MRD is available and 10 patients have reached MRD negativity. Response quality was not associated with risk factors, age or with lenalidomide dose in those receiving 6 cycles of treatment. Remarkably, one of three patients with deletion 17 achieved an MRD negative CR. Since we could not define a clinical predictor for the patients' tolerance of lenalidomide, we performed extensive immunophenotyping of T cells in pretreatment samples, using markers for functional T cells subsets, their Th polarity and for suppressive or exhausted T cell subsets. Employing a combined endpoint including non-hematological dose-limiting events or MTD 〈 10mg as a comparator, we identified a fraction of non-exhausted memory CD4 cells as highly significant predictor of dose-limiting non-hematologic events (p

Description: Rhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by twoRHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III, was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3′ breakpoints of all knownDVItypes shown to be distinct. We differentiated the 5′ breakpoints of DVItypeI andDVItype II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface ofDVItype III was normal (about 12,000 antigens/cell; DVItypeI, 500;DVItype II, 2,400) based on the determination of an RhD epitope density profile. DVItype II and DVItype III occurred as CDe haplotypes, and DVItype I as a cDE haplotype.The distribution of the DVItypes varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.

Description: Abstract 1773 T cell exhaustion is a state of T cell dysfunction in response to chronic antigen exposure, marked by impaired effector function and the continued expression of inhibitory receptors such as Programmed Death 1 (PD-1) (Wherry EJ. Nat Immunol. 2011 Jun;12(6):492–9.). Because tumour growth in chronic lymphocytic leukaemia (CLL) occurs over a long period of time, we hypothesized that the continued exposure of T cells to a CLL-derived antigen could also lead to a state of T cell exhaustion. We therefore investigated whether T cell exhaustion is induced in CLL by using the Eμ-TCL1 transgenic (tcl1tg) tumour transfer mouse model for this disease (Hofbauer JP, et al. Leukemia. 2011 Sep;25(9):1452-8) and by analyzing primary samples from CLL patients. We found that the number of PD-1+ T cells was increased in both CD4+ and CD8+ populations and in all lymphoid compartments examined of the Eμ-TCL1 transgenic (tcl1tg) tumour recipient mice, but not in recipient mice receiving wildtype (WT) splenocytes showing that leukemic mice have an increased number of T cells displaying an exhausted phenotype that is induced by the presence of CLL cells. We next assessed the expression of the ligands for PD-1 on the surface of murine CLL cells. Peripheral CLL tumour cells showed only a modest increase in PD-L1 expression as compared to WT B cells. However, lymph node and spleen residing tumour cells showed a marked increase in PD-L1 expression, which suggests a microenvironment-induced upregulation of PD-L1 on tumour cells, e.g. by their close contact to accessory cells. To validate our results on primary human CLL samples, we collected peripheral blood from 89 unselected CLL patients and 18 healthy donors and observed an increase in surface expression of PD-1 on the CD4+ and CD8+ T cell populations. While the percentage of PD-1+ CD4+ T cells in chemonaive patients was comparable to healthy donors, chemotherapy drastically increased the number of PD-1-expressing CD4+ T cells (63.81% ±19.75 vs 35.70% ±19.22; p

Description: A Rhesus D (RhD) red blood cell phenotype with a weak expression of the D antigen occurs in 0.2% to 1% of whites and is called weak D, formerly Du. Red blood cells of weak D phenotype have a much reduced number of presumably complete D antigens that were repeatedly reported to carry the amino acid sequence of the regular RhD protein. The molecular cause of weak D was unknown. To evaluate the molecular cause of weak D, we devised a method to sequence all 10RHD exons. Among weak D samples, we found a total of 16 different molecular weak D types plus two alleles characteristic of partial D. The amino acid substitutions of weak D types were located in intracellular and transmembraneous protein segments and clustered in four regions of the protein (amino acid positions 2 to 13, around 149, 179 to 225, and 267 to 397). Based on sequencing, polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction using sequence-specific priming, none of 161 weak D samples investigated showed a normal RHD exon sequence. We concluded, that in contrast to the current published dogma most, if not all, weak D phenotypes carry altered RhD proteins, suggesting a causal relationship. Our results showed means to specifically detect and to classify weak D. The genotyping of weak D may guide Rhesus negative transfusion policy for such molecular weak D types that were prone to develop anti-D.

Description: Antigen D compatible transfusion is standard practice in modern transfusion therapy, warranting proper antigen D typing in all blood donors. Blood group genotyping is increasingly utilized for prenatal diagnosis and after recent transfusions. RHD genotyping is of practical importance to overcome the limitations of standard serology, which frequently fails to detect some weak D and chimeric red blood cell (RBC) populations, and to enhance clinical safety for blood transfusion recipients. Recently, the transfusions of blood units from chimeric donors were reported to having induced an acute transfusion reaction and multiple anti-D immunizations. The latter donor escaped the serologic detection in 13 donations but was uncovered by the strategy reported in this study. Since January 2002 all D negative (D neg.) first time donors at blood center A were tested for carrying the RHD gene. We evaluated the results of this two years’ routine testing and compared them to a screening study conducted in the independent blood center B. This approach contributed to define the utility of RHD genotyping for the quality control of D neg. RBC units. In two independent blood centers we examined 9,931 and 5,115 serologic D neg. blood donors. Samples were tested in pools of 20 donors by PCR-SSP for RHD intron 4 or for RHD exon 4, exon 7 and exon 10. 21 RHD positive donors were detected in center A and 18 RHD positive donors in center B among the serologic D neg. donors. The molecular bases of the RHD positive samples were resolved in all cases. A total of 10 RHD alleles were novel: RHD(T201R, F223V, P291R) dubbed weak D type 4.3, RHD(I374N) dubbed weak D type 32, RHD(del147), RHD(del343), RHD(del449), RHD(del785), RHD(L153P), RHD(Y269X), RHD(IVS3+2T〉A) and RHcE(1–3)-D(4–10). 13 samples in center A represented 5 known RHD alleles, most often RHDψ (n = 5), RHD(IVS3+1G〉A) (n = 4) and RHD(M295I) in CDe (n = 2); 7 samples in center B represented the 3 known RHD alleles RHD(IVS3+1G〉A) (n = 4), RHD-CE(2–9)-D2 (n = 2) and RHD(M295I) (n = 1). 9 donors in center A represented Del; in center B 9 were weak D and 6 Del; 13 of the remaining donors were confirmed to be D neg. despite carrying the RHD gene. We concluded that RHD genotyping of serologic D neg. donors at two facilities revealed carriers of the RHD gene expressing antigen D, albeit at low levels, in the range of up to 1:1,000 and 1:350 donors, respectively. At least 24 donors carried RHD alleles that were known or shown to express a weak D or Del phenotype. The RBC units donated by these donors may be capable of causing at least secondary anti-D immunization. This possible adverse clinical outcome was avoided by RHD genotyping each donor only once. Use of RHD genotyping would obviate the need to tightly control the sensitivity of serologic anti-D testing in blood donors. Further studies are needed to corroborate the current experience in particular in donors of non-white ethnic background. However, we think that RHD genotyping in first time blood donors has the potential to become a routine procedure in blood centers.

Description: Clonal hematopoiesis (CH) is associated with age and an increased risk of myeloid malignancies, cardiovascular risk, and all-cause mortality. We tested for CH in a setting where hematopoietic stem cells (HSCs) of the same individual are exposed to different degrees of proliferative stress and environments, ie, in long-term survivors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their respective related donors (n = 42 donor-recipient pairs). With a median follow-up time since allo-HSCT of 16 years (range, 10-32 years), we found a total of 35 mutations in 23 out of 84 (27.4%) study participants. Ten out of 42 donors (23.8%) and 13 out of 42 recipients (31%) had CH. CH was associated with older donor and recipient age. We identified 5 cases of donor-engrafted CH, with 1 case progressing into myelodysplastic syndrome in both donor and recipient. Four out of 5 cases showed increased clone size in recipients compared with donors. We further characterized the hematopoietic system in individuals with CH as follows: (1) CH was consistently present in myeloid cells but varied in penetrance in B and T cells; (2) colony-forming units (CFUs) revealed clonal evolution or multiple independent clones in individuals with multiple CH mutations; and (3) telomere shortening determined in granulocytes suggested ∼20 years of added proliferative history of HSCs in recipients compared with their donors, with telomere length in CH vs non-CH CFUs showing varying patterns. This study provides insight into the long-term behavior of the same human HSCs and respective CH development under different proliferative conditions.

Description: Rhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by twoRHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III, was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3′ breakpoints of all knownDVItypes shown to be distinct. We differentiated the 5′ breakpoints of DVItypeI andDVItype II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface ofDVItype III was normal (about 12,000 antigens/cell; DVItypeI, 500;DVItype II, 2,400) based on the determination of an RhD epitope density profile. DVItype II and DVItype III occurred as CDe haplotypes, and DVItype I as a cDE haplotype.The distribution of the DVItypes varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.

Description: Introduction Lenalidomide has shown encouraging activity in monotherapy trials in CLL, but tumor lysis and tumor flare presented obstacles in development. We and others previously presented first data on combinations of lenalidomide with standard treatment regimens in CLL. As reported at ASH 2011 we combined Lenalidomide safely and efficaciously with the combination of Fludarabine and Rituximab, achieving early reduction of tumor load without tumor lysis or tumor flare and with high response rates. We also uncovered a patient population unable to tolerate higher Lenalidomide doses and marked by an exhausted T cell subset, measured pre-treatment. We now report final results of this trial, including the maintenance phase. Study design In induction Lenalidomide was combined with Fludarabine (40mg/m2 po d1-3 q28d) and Rituximab (375mg/m2 iv d4 cycle 1; 500mg/m2 iv d1 cycles 2-6, q28d). In cycle 1 Lenalidomide was added day 7-21 at 2.5 mg. Toxicity permitting, Lenalidomide dose was escalated to 5, 10, 15, 20 and 25mg day 1-21 over cycles 2-6. Subsequent maintenance treatment was two-monthly Rituximab at 375mg/m2 and Lenalidomide at the last dose tolerated in combination in a 28 day cycle without interruption for 6 months. The main goal of this treatment phase was to establish safety and efficacy as a secondary endpoint to the study. Results Patient characteristics of 45 recruited patients were previously reported: median age was 66 years and at least one molecular high risk feature was present in 64% of patients. No systematic toxicity determining an MTD in induction, the primary study endpoint, was found. The median daily dose in cycle 6 was 15mg in 40 evaluable patients, with 3 patients receiving the last cycle without lenalidomide. Toxicity and efficacy of the induction regimen were reported previously. Maintenance treatment was started in all 40 patients finishing induction. Three patients that finished without lenalidomide received only Rituximab. The median starting dose for all 40 patients was 15mg daily and 70% started with 10mg upwards. In total, 46% needed dose reductions, with prolonged neutropenia being the main reason, but 47% received doses above 10mg up to cycle 6 of maintenance. Interestingly, 9/13 patients receiving 25mg as maintenance were able to receive the treatment uninterrupted for 6 months, suggesting that a biologically select group may tolerate very high doses. As alluded to the major toxicity was neutropenia with 45% and 27% reaching G3 and G4, respectively. Surprisingly this did not translate into a relevant signal for infections. Grade 3, but no G4 infections, were observed in 5% of patients and all other G3/4 toxicities remained below 5%. Compared to the reported incidence of skin reactions in induction, we did not observe a significant signal in the maintenance phase. Improvement of response from PR after induction to CR at the end of maintenance was observed in 25%. The overall best response in ITT to the regimen during induction and maintenance was CR in 67% and PR in 29%. Median follow up of the study is now 35 months, at which point PFS is 89% and observed median PFS is currently 46 months. Exploratory analyses show no significant influences of age〉65, mutation state or CD38 risk on PFS, but undetectable MRD after induction and high risk cytogenetics showed borderline effects (both p=0.08), the latter (2 cases with del17p and 9 with del11q) being driven by relapses in patients with del11q with a median PFS of 42 months in this group. Conclusions A combination of Lenalidomide with FR followed my maintenance with Lenalidomide and Rituximab proved clinically feasible. While initial dose-finding was complicated by highly individual levels of tolerance to lenalidomide in the combination (with skin toxicity being a major problem), the main toxicity in maintenance was neutropenia. Although the important myelotoxicity did not translate into a rate of infection above that expected after induction treatment, our judgment is, that a more moderate approach to dosing may be warranted in maintenance, since a majority of patients had to be dose-adjusted. Finally, the PFS observed with this induction and maintenance regimen seems encouraging by comparison with other first line regimens. While the exploratory nature of the trial clearly limits this conclusion, we further explore combination approaches with lenalidomide. Disclosures: Egle: Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Research Funding. Off Label Use: Lenalidomide in CLL. Pleyer:Celgene: Honoraria, Research Funding. Fridrik:Roche: Honoraria. Thaler:Roche: Honoraria, Research Funding. Greil:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding.

Description: Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.