ALBERT

Beschreibung: The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is biased and characterized by the existence of subsets of cases with closely homologous (“stereotyped”) complementarity-determining region 3 (CDR3) sequences. In the present series, 201 (21.9%) of 916 patients with CLL expressed IGHV genes that belonged to 1 of 48 different subsets of sequences with stereotyped heavy chain (H) CDR3. Twenty-six subsets comprised 3 or more sequences and were considered “confirmed.” The remaining subsets comprised pairs of sequences and were considered “potential”; public database CLL sequences were found to be members of 9 of 22 “potential” subsets, thereby allowing us to consider them also “confirmed.” The chance of belonging to a subset exceeded 35% for unmutated or selected IGHV genes (eg, IGHV1-69/3-21/4-39). Comparison to non-CLL public database sequences showed that HCDR3 restriction is “CLL-related.” CLL cases with selected stereotyped immunoglobulins (IGs) were also found to share unique biologic and clinical features. In particular, cases expressing stereotyped IGHV4-39/IGKV1-39-1D-39 and IGHV4-34/IGKV2-30 were always IgG-switched. In addition, IGHV4-34/IGKV2-30 patients were younger and followed a strikingly indolent disease, contrasting other patients (eg, those expressing IGHV3-21/IGLV3-21) who experienced an aggressive disease, regardless of IGHV mutations. These findings suggest that a particular antigen-binding site can be critical in determining the clinical features and outcome for at least some CLL patients.

Beschreibung: High-dose therapy plus autologous stem cell transplant (ASCT) is the standard of care for patients with multiple myeloma (MM) aged ≤65 years. Melphalan–prednisone (MP)-based therapy is the standard for non-ASCT candidates but is not typically used for transplant-eligible patients as prolonged therapy with melphalan can adversely affect stem cell collection. The phase 3 VISTA study demonstrated the superior efficacy of bortezomib plus MP (VMP) versus MP in previously untreated MM patients ineligible for ASCT. In this phase 2 study, we evaluated the efficacy of a shorter course of VMP on a different treatment schedule as induction therapy prior to ASCT or as frontline therapy in non- ASCT candidates. Patients aged ≥18 years with previously untreated MM received up to six 28-day cycles of bortezomib 1.3 mg/m2 IV, days 1, 4, 8, and 11, plus oral melphalan 6 mg/m2 and oral prednisone 60 mg/m2, days 1–7. After 2–6 cycles, ASCT-eligible patients could proceed to stem cell mobilization (G-CSF 10 mg/kg/day ± GM-CSF 250 mg/m2/ day or cyclophosphamide 4 g/m2 + GM-CSF) and conditioning with melphalan 200 mg/ m2 (140 mg/m2 if aged 〉65 years). Response was assessed every two cycles and post- ASCT by International Uniform Response Criteria. The primary end point was complete response (CR) rate to VMP. A total of 45 patients were enrolled; 27 were male. Median age was 63 years (range 33–75). MM subtype was 67% IgG, 16% IgA, and 9% each κ- and λ- light-chain; 37% of patients had ISS Stage III MM, 22% had ECOG performance status 〉1, and 70% had ≥40% plasma cells in bone marrow. In total, 20 patients proceeded to ASCT. Median duration of VMP was 4 cycles in both non-ASCT (range 1–6) and ASCT (range 2–6) patients. Response rate (best response) to VMP was 95% (42 of 44 evaluable patients), including 9% stringent CR (sCR), 9% CR (18% ≥CR [95% CI: 7%, 30%]), 27% very good partial response (VGPR), and 50% partial response (PR). Best response was achieved after cycle 2 in 10 patients, cycle 4 in 25 patients, and cycle 6 in 7 patients. All 20 ASCT patients had successful stem cell mobilization; median yield of CD34+ cells/ kg was 5.6 x 106 (range 2.3–12.2 x 106), in a median of 2 collection days. Post-transplant responses were 10% sCR, 20% CR, 55% VGPR, and 5% PR; the remaining 2 patients need further follow-up for response assessment. Response improved post-VMP to post-ASCT in 10 patients (6 PR to VGPR, 2 PR to CR, 2 VGPR to CR). After median follow-up of 14.0 months (range 7.4–47.7) and 14.6 months (range 8.2–42.9) in non-ASCT and ASCT patients, respectively, both median time to progression and progression-free survival were 19.8 months (95% CI: 14.3 months, not estimable [NE]) in non-ASCT patients and 27.9 months (95% CI: 14.6 months, NE) in ASCT patients. A total of 7 patients (5 non- ASCT, 2 ASCT) have died; 1-year survival rate was 82% (95% CI: 59%, 93%) in non- ASCT patients and 95% (95% CI: 69%, 99%) in ASCT patients. Most common grade 3/4 adverse events in all 45 patients during VMP therapy included peripheral neuropathy (24%), thrombocytopenia (20%), neutropenia (18%), and infection (9%). Only 1 patient had deep-vein thrombosis. In conclusion, VMP represents a highly effective therapy for previously untreated MM, with 45% of patients achieving VGPR or better, including 18% sCR/CR. Toxicities were predictable and generally manageable. Short-course VMP therapy did not negatively impact stem cell mobilization, supporting its use as induction therapy prior to ASCT. Very high post-transplant response rates were seen, with 85% of patients achieving ≥VGPR, including 30% sCR/CR. Since achievement of CR/VGPR is associated with improved long-term outcomes in MM, the preliminary outcome data presented here appear promising; however, longer follow-up is required.

Beschreibung: Factor VII (FVII) consists of an N-terminal γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. The complex of tissue factor (TF) and FVIIa activates FIX and FX during coagulation. FVIIa on its own is structurally more “zymogen-like” and when bound to TF it is more “active enzyme-like.” We obtained crystal structures of EGR-VIIa/soluble (s) TF (2.9 Å resolution), dansyl-EGR-VIIa/sTF (1.9 Å resolution) and benzamidine-VIIa/sTF (1.6 Å resolution). We also investigated the effect of TF binding on the S1, S2, and S3/S4 subsites (Schechter and Berger, BBRC, 27:157-162, 1967) in FVIIa. The affinity of variously inhibited FVIIa to sTF was also measured using Biacore technology. For obtaining second order inhibition rate constants, FVIIa ± soluble (s) TF was incubated with each inhibitor for various times, diluted several fold and assayed for the residual FVIIa activity. The second order rate constants were obtained by plotting the first order rate constants versus the inhibitor concentrations. These data are summarized in the table below. From these data it appears that all subsites are affected upon FVIIa binding to sTF. Since in the crystal structure of EGR-VIIa/sTF the P1 Arg residue is the only residue that makes contact with FVIIa, it follows that the S1 site is affected ~10-fold upon binding to sTF. Adding a dansyl group that partially occupies the S3/S4 position (1.9 Å structure) increases the second order rate constant 7-fold (2.41 versus 0.35) over that of EGR-ck. Moreover the addition of Pro (DFPR-ck) or Phe (DFFR-ck) residue occupying the S2 position increases the second order rate constant 357-fold and 1500- fold, respectively (125 and 525 versus 0.35). Thus, comparison of dEGR, DFPR, DFFR inhibition suggests that FVIIa prefers Phe at S2 and at S3/S4 positions, and that TF opens up the S1/S2/S3/S4 sites for substrate or inhibitor occupancy. These data are consistent with LTR (P3/P2/P1) residues in FX at its activation cleavage site as well as with LTR (P3/P2/P1) residues and FTR (P3/P2/P1) residues at the 145-146 and 180-181 FIX activation cleavage sites, respectively. Thus, these studies with chloromethylketone inhibitors have biologic relevance. For Biacore studies, sTF was amine coupled to a CM5 chip. The binding of unoccupied active site FVIIa in 5 mM calcium to sTF was characterized by a KD of 7 nM. Benzamidine (10 mM)-VIIa, p-aminobenzamidine (pAB, 1 mM)-VIIa, EGR-VIIa, dEGR-VIIa, DFPR-VIIa and DFFR-VIIa each bound to sTF with KD values ranging from 1- 2 nM. These affinity measurements indicate that the S1 site occupied FVIIa molecule (benzamidine-FVIIa, pAB-VIIa) has essentially the same conformation as the S1/S2/S3/S4 occupied FVIIa. This conclusion is consistent with similar crystal structures of variously inhibited FVIIa molecules complexed with sTF. The differential rates of incorporation of various chloromethylketone inhibitors could be due to the interaction of various residues (P1, P2, P3, P4) with the corresponding active subsites (S1/S2/S3/S4) of FVIIa. Additionally, the rate of incorporation of chloromethylketone inhibitors into FVIIa also involves the irreversible alkylation step, which could be faster for DFFR-ck and DFPR-ck. Once these inhibitors are incorporated, it appears that they induce the same conformation in FVIIa as achieved by S1 site occupancy alone. Thus S1 site occupancy in FVIIa induces the required conformation to modestly increase the affinity for TF. Second Order Rate Constants for Inhibition of FVIIa ± sTF with Various Chloromethylketone (ck) Inhibitors Inhibitor Minus sTF k (min−1 mM−1) Plus sTF k (min−1 mM−1) Fold Difference EGR-ck 0.04 0.35 8.8 dansyl EGR-ck 0.07 2.41 34.4 (D)FPR-ck 2.3 125 54.3 D)FFR-ck 5.6 525 93.8

Beschreibung: Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)–induced Ank-1–null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in β-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

Beschreibung: Relapse following remission induction chemotherapy remains a barrier to survival in approximately 20% of children suffering from acute lymphoblastic leukemia (ALL). To investigate the mechanism of relapse, 27 matched diagnosis and relapse ALL samples were analyzed for clonal populations using polymerase chain reaction (PCR)–based detection of multiple antigen receptor gene rearrangements. These clonal markers revealed the emergence of apparently new populations at relapse in 13 patients. More sensitive clone-specific PCR revealed that, in 8 cases, these “relapse clones” were present at diagnosis and a significant relationship existed between presence of the relapse clone at diagnosis and time to first relapse (P 〈 .007). Furthermore, in cases where the relapse clone could be quantified, time to first relapse was dependent on the amount of the relapse clone at diagnosis (r = −0.84; P = .018). This observation, together with demonstrated differential chemosensitivity between subclones at diagnosis, argues against therapy-induced acquired resistance as the mechanism of relapse in the informative patients. Instead these data indicate that relapse in ALL patients may commonly involve selection of a minor intrinsically resistant subclone that is undetectable by routine PCR-based methods. Relapse prediction may be improved with strategies to detect minor potentially resistant subclones early during treatment, hence allowing intensification of therapy.

Beschreibung: Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).

Beschreibung: Abstract 3315 Poster Board III-203 Background Late-onset noninfectious pulmonary complications (LONIPC) are both frequent and severe complications of allogeneic hematopoietic stem cell transplantation (HSCT). They include bronchiolitis obliterans (BO), interstitial pneumonitis (IP) and bronchiolitis obliterans with organizing pneumonia (BOOP). Their outcome is usually unfavourable. Early diagnosis and treatment may improve the prognosis. We assessed the feasibility and benefits of home surveillance of pulmonary function for early diagnosis of LONIPC in allogeneic HSCT recipients. Patients and methods This prospective study included all patients with a landline telephone living in Paris area. Monitoring with a portable spirometer (spirotel®, M-Elect France) was scheduled to start 3 months after transplant and has been performed for 18 months, or longer if respiratory failure occured. Vital capacity (VC), forced expiratory volume per second (FEV1) and mid expiratory flow 25 to 75 values (FEF25-75) were measured. Data was transmitted by phone to hospital twice a week. If significant deterioration occurred, defined by at least a 20% drop, spirometry and plethysmography were performed, and if alterations of pulmonary functions were confirmed, further investigations (thorax CT-scan and fiberoptic bronchoscopy with broncho-alveolar lavage.) were performed in the pneumology unit. Results Between June 2001 and November 2008, 336 patients received a HSCT in the hematology unit of Pitié-Salpêtrière Hospital. One hundred and ninety one patients were included in the study before transplant. One hundred and twenty patients were actually equipped after transplant, whereas 71 were excluded because of poor clinical status or early death. Median age of the 120 equipped patients was 46 (20-66) years. HSCT was performed with HLA sibling donor in 52.5% and myeloablative conditioning regimen in 60%. Acute graft-versus-host disease (GVHD) grade II-IV occurred in 44% patients and chronic GVHD in 57%. During monitoring, 32 confirmed telemetric deteriorations occurred in 25 patients at a median time of 15 months post-transplant (3-61). They allowed the diagnosis of LONIPC in 12 patients (6 BO, 1 IP, 4 patients presented both BO and IP (BO-IP)). The thirtheen remaining patients presented infection (N=5), cardiac failure (N=3), asthma (N=3) or others (N=2). During, the whole follow-up, 28 patients presented LONIPC (16 BO, 5 IP, 7 BO-IP). Twelve of them were detected by telemetric spirometry, whereas LONIPC occurred before the onset or after the end of the monitoring in 11 patients. In 3 cases of IP, 1 case of BO and 1 case of BO-IP, the diagnosis of LONIPC was not performed by telemetric monitoring. Treatment consisted in introduction or increasing of systemic immunosuppressive therapy for 23/28 patients, the 5 remaining patients were exclusively treated with azithromycin and/or inhaled steroids and long acting β2 mimetics. At the last follow up (25±20 months post-transplant), lung functional improvement (defined as improvement of FEV1〉200mL) was observed in 54% patients, functional deterioration (defined as a decreasing of the FEV1〉 200mL) was observed in 14% patients, and functional stabilization in 32%. Functional improvement occurred in 80% and 86% in patients with IP and BO-IP respectively, and in 31% patients with BO. During follow-up no death related to LONIPC occurred Conclusion Home telemetric surveillance allowed an early detection of LONIPC. With precocious diagnosis and treatment, no death related to LONIPC was observed, and a functional improvement occurred for 54% patients, especially those suffering IP or BO-IP. Disclosures No relevant conflicts of interest to declare.