ALBERT

Description: Background Diagnosis and prognosis of patients with established MDS/CMML is currently based on WHO criteria and the IPSS prognostic Index. High variation of survival in subgroups warrants the search for additional criteria. A comparison of CFU-C cultures with WHO criteria and IPSS score has not yet been done in a large patient group. Patients and methods We analyzed in a single center retrospective cohort study 93 untreated consecutive patients (55 male/ 38 female; median age: 66 years; range: 13 – 88 years) admitted between July 1992 and June 2002 and diagnosed as MDS/CMML (RA/RARS(4), MDS-U (2), RCMD (26), RAEB I/II (44) and CMML I/II (17). All patients had an unequivocal diagnosis of MDS or CMML according to WHO criteria. Simultaneous examinations of blood, bone marrow (cytology and biopsy), BM-cytogenetics and BM-and PB cultures for CFU-GM and BFU-E were done. Culture results were scored blindly and classified either as “Low risk CFU-C“ including normal growth (N=2), no colony growth (N=6) or reduced growth of normal colonies (N=19) or as „High risk CFU-C’s“ including excess normal growth in PB termed “MPS pattern” (N=5), discrete leukemic cluster growth (N=22), abundant leukemic cluster growth (N=14) or the „CMML pattern“ defined as giant“ pseudonormal“ CFU-GM and strongly reduced BFU-E (N=23). Culture score was compared with the WHO diagnosis and with the IPSS score in all 93 patients and survival was assesed in 82 patients treated with conventional therapy (12 allografted patients were excluded) after minimal observation time of 3 years in July 2005. Results Comparison of WHO diagnosis with culture score Low risk CFU-C score High risk CFU-C score RA/RARS/RCMD/MDS-U 15 16 RAEBI/II 12 30 CMML-I/II 0 17 The typical CMML pattern was observed in 13 of 17 patients with CMML (Specificity 94%). Comparison of IPSS Score with culture score low risk CFU-C score low risk CFU-C score High risk CFU-C score High risk CFU-C score IPSS Score alive/dead mean survival(d) alive/dead mean survival(d) Low and Int-1 4/4 2022+/−543 8/39 965+/−143 Int-2 and High 1/1 1024+/−701 1/26 565+/−92 Mean survival time for patients with a low/intermediate-1 IPSS score was less than half if they had a high risk CFU-score (p

Description: The donor/recipient pair provides a model for evaluating telomere shortening and cell senescence in a transplant setting, since cells have the same origin but a different fate. High replicative stress on stem cells accelerates telomere shortening in all leukocytes within the first year after allogeneic hematopoietic stem cell transplantation (HSCT). Thereafter, telomere length dynamics of HSCT-recipients appear not to differ from their donors. We evaluated telomere shortening in different leukocyte subsets in very long survivors after HSCT and looked for associations with transplant events. This was a prospective, cross-sectional study in which we analyzed in a blinded way the telomere length by automated multicolor flow-FISH in 20 patients and their respective HLA-matched sibling donors. The median age at study time was 41 years for the recipients (range 5–50) and the donors (range 3–45), 65% of the patients were male. The donor was younger in 60% and a female in 35%. The median follow-up after HSCT was 17.5 years (range 12–25). Two patients (10%) received HSCT for severe aplastic anemia and 18 (90%) for a hematological malignancy. Total body irradiation (TBI) was part of the conditioning in 85% of the patients and all received bone marrow as source of stem cells. Acute graft versus host disease (GVHD) was observed in 55% of the patients and chronic GVHD in 40%. The age-matched and cell-type-specific absolute telomere length values for the recipients and the donors fell between the 1st and 99th percentile of the normal distribution. The telomere length (mean ± std) in recipients as compared to donors was significantly shorter (p 〈 0.01) for granulocytes (6.7 kb ± 1.0 vs 7.2 ± 0.9), CD45RA-positive T-cells (5.6 kb ± 1.2 vs 6.3 ± 1.2); memory T-cells (5.1 kb ± 0.9 vs 5.6 ± 0.9), B-cells (7.2 kb ± 1.2 vs 7.9 ± 1.1) and NK/NKT-cells (5.1 kb ± 0.8 vs 5.6 ± 1.5). The mean difference in telomere length between the pairs was in the range of 0.5–0.7 kb for each subset of leukocytes; based on the telomere length values we could predict donors and recipients in a 100 %. Age, sex, diseases, conditioning with fractionated or non-fractionated TBI and acute GVHD had no impact on relative telomere loss. Patients with chronic GVHD (n=8; p=0.04) and particularly those with extensive chronic GVHD (n=5; p=0.004) presented with significant telomere shortening in CD45RA-positive T-cells. In recipients with extensive chronic GVHD telomere shortening was also found in B-cells (p=0.04). For leukocytes there was a trend in telomere shortening over time since HSCT (p=0.06). Our results confirm and extend previous findings that peripheral blood cells have shorter telomeres in long-term recipients after allogeneic HSCT. The difference in telomere length compared to the telomere length value in the respective donor corresponds to approximately 10–15 years of cell aging. In patients with chronic GVHD with more pronounced telomere shortening in certain subsets of lymphocytes but not in granulocytes, this difference corresponds to approximately 40–60 years of cell aging. These findings are compatible with a concept of chronic GVHD as a disease of disturbed immunity and raise the question of immuno-senescence or late altered immunity in chronic GVHD.

Description: An acquired gain-of-function mutation in the Janus kinase 2 (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPDs). To test the hypothesis that JAK2-V617F is the disease-initiating mutation, we examined whether all cells of clonal origin carry the JAK2-V617F mutation. Using allele-specific polymerase chain reaction (PCR) assays for the JAK2 mutation and for the X-chromosomal clonality markers IDS and MPP1, we found that the percentage of granulocytes and platelets with JAK2-V617F was often markedly lower than the percentage of clonal granulocytes determined by IDS or MPP1 clonality assays in female patients. Using deletions of chromosome 20q (del20q) as an autosomal, X-chromosome–independent clonality marker, we found a similar discrepancy between the percentage of cells carrying JAK2-V617F and del20q. Our results suggest that in a proportion of patients with MPDs, JAK2-V617F occurs on the background of clonal hematopoiesis caused by a somatic mutation in an as-yet-unknown gene.

Description: The International Unrelated Search and Transplant (IMUST) Study included 42 European centers and prospectively accrued 345 unrelated donor (URD) and 699 matched sibling donor (MSD) SCT between 1989 and 19921. We now compare the incidence and severity of late events occurring in the 108 URD and 355 MSD recipients surviving more than 2 years, range 2.0–13.9 years after SCT. In this late surviving cohort 11/108 URD and 37/355 MSD recipients were transplanted for non-malignant disease, and 97/108 URD and 318/355 MSD recipients for haematological malignancy. In the URD cohort 85 recipients received TBI conditioning and 33 T-cell depletion. In the MSD cohort 259 received TBI conditioning and 68 T-cell depletion. In patients surviving more than 2 years after SCT the Karnofsky score was 90–100 in 262, 80 or less in 104 and unknown in 77. The Karnofsky score was similar in the URD and MSD SCT cohorts. One hundred and five late deaths occurred amongst the 463 patients surviving more than 2 years. Deaths were relapse related in 60 cases, transplant related in 29, due to second malignancy in 4, other causes in 6 and unknown causes in 6. Extensive CGVHD was present in 84 recipients surviving more than 2 years, limited CGVHD in 179 and no CGVHD in 200. The cumulative incidence (CI) for survival at 12 years in patients surviving more than 2 years with CGVHD was 49% (44–55) in the MSD cohort and 59% (51–70) in the URD cohort. The CI for extensive CGVHD was slightly higher in the URD than MSD recipients, 49% (44–55) versus 30% (22–42), p=0.1. Twelve year survival was 77+/−5% for the MSD and 67+/−11% for the URD cohort, p=0.1. For patients surviving more than 2 years with extensive CGVHD subsquent survival at 12 years was poor at 49+/−14%, compared with 80+/−7% for limited CGVHD, and 82+/−6% for no CGVHD, p=0.0001. In the presence of extensive CGVHD 12 year survival was similar after MSD 43+/−20% and URD transplants 51+/−20%, p=0.89. A multivariate Cox regression model showed CGVHD was the only variable significantly associated with survival beyond 2 years, p=

Description: Mutations in exon 12 of the JAK2 gene have been identified in a minority of patients with myeloproliferative disorders (MPD) and are associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis. We compared the lineage distribution of JAK2 mutations in the peripheral blood of 8 PV patients with mutations in exon 12 and 21 PV patients with the JAK2-V617F mutation. Five different exon 12 mutations were observed in the 8 patients studied. Peripheral blood cells were fractionated to obtain granulocytes, platelets and mononuclear cells, which were further separated by FACS into T cells, B cells, NK cells and monocytes. Using a sensitive and quantitative assay to assess the percentages of chromosomes carrying exon 12 mutations, we detected exon 12 mutations in purified granulocytes, monocytes and platelets of all patients studied. A similar distribution was also found for the JAK2-V617F mutation. Exon 12 mutation was absent in sorted lymphoid cells of 5/8 patients, in 2/8 patients only NK cells were positive and in 1/8 patients also B cells carried the mutation. Exon 12 mutations were absent in T cells of all patients studied and JAK2-V617F was present in T cells of only 1/21 patients. Thus, inter-individual differences are notable in the involvement of lymphoid lineages for both exon 12 and JAK2-V617F mutations. To determine the presence of the JAK2 mutations in the erythroid lineage, we performed colony assays in methylcellulose, picked single erythroid colonies grown in the presence or absence of Epo and determined the allelic ratios for each individual colony. In 4/8 patients an exon 12 mutation (E543-D544del) was present in all EECs examined and similarly, in 8/12 patients the JAK2-V617F mutation was found in all EECs. In the remaining patients, we detected some EECs with only the wild type JAK2, suggesting that additional clonal events may also be present in some patients with exon 12 mutations. Interestingly, one patient carried exon 12 and JAK2-V617F mutations. None of the erythroid colonies in this patient carried both mutations simultaneously, indicating that the exon 12 mutation and JAK2-V617F represent two separate clones. One patient displayed erythroid colonies homozygous for the exon 12 mutation and an allelic ratio greater that 50% in granulocytes, indicating that progression to homozygosity can occur in some patients. The lineage distributions of exon 12 mutations and JAK2-V617F are similar and do not explain why exon 12 mutations are associated solely with PV phenotype, whereas JAK2-V617F can also cause essential thrombocythemia or primary myelofibrosis.

Description: Nursing in LAF units and routine high dose IVIG were considered essential elements and standard of care in allogeneic HSCT during the 1980s. They were abandoned by many teams in view of high costs, lack of formal proof of evidence and improved diagnostic and therapeutic interventions against viral and fungal infections. A recent prospective study showed no benefit of routine IVIG but few data have analysed the consequences of the combined change in practice. We made these changes at our institution in two stages during a 10-year period: change from care in LAF rooms to single rooms (SR) and change from routine high dose IVIG to targeted IVIG replacement according to CDC guidelines. Basic strategies in supportive care and data collection remained unchanged. This resulted in a retrospective analysis of 357 allogeneic HSCT in three cohorts, i.e. LAF + IVIG, SR + IVIG and SR − IVIG (Table). Endpoints analysed were survival, transplant related mortality (TRM), relapse rate, incidence and severity of GvHD and infectious complication rates defined as: sepsis, septic thrombosis, urinary tract infections, pneumonias, and CMV reactivation. Results were adjusted by multivariate analyses for all the changes over time (see Table *significant difference between groups). There were more patients in advanced stage of disease and with higher age in more recent years. There were significantly fewer septicemias per hospital day but not per day in neutropenia in the LAF + IVIG group without impact on survival. There were no significant differences in any of the other outcomes analysed. Abandoning LAF and routine high dose IVIG use did not impact negatively on HSCT outcome. These data support current practice. Group LAF + IVIG SR + IVIG SR, no IVIG Total N 116 134 107 357 Male 55% 51% 62% 59% Age, years* 34 34 40 35 Median, range 4–58 2–62 1–62 35 Diagnosis* Leukemias 85% 83% 82% 84% Lymphoproliferative 7% 13% 15% 11% Others 8% 4% 3% 5% Early disease* 46% 38% 29% – Late disease 54% 72% 71% 66% % RIC* 0% 10% 32% – Days in hospital* 53 32 28 38 Days in neutropenia* 16 15 11 14 Day 100 TRM 18% 20% 22% 20%

Description: An acquired somatic mutation in the JAK2 gene (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPD). In most studies the JAK2-V617F mutation has been analyzed at single time point. Here we performed a retrospective single center study on 73 MPD patients (36 polycythemia vera (PV), 29 essential thrombocythemia (ET), and 8 primary myelofibrosis (PMF)) from whom at least two blood samples (mean=5, range 2–17) were available with an interval of at least 8 months. The mean follow-up period was 35±17 months (range 8–78 months). The allelic ratio of JAK2-V617F (%T) was determined in DNA from purified peripheral blood granulocytes by allele-specific PCR. In 73 MPD patients studied, 53 (73%) carried the JAK2-V617F mutation (32 PV, 18 ET, 3 PMF). None of the 20 patients negative for JAK2-V617F acquired the JAK2-V617F mutation during the observation period (n=20, mean number of samples=4). In the majority of the JAK2-V617F positive patients (35/53; 66%) the JAK2-V617F allelic ratios remained remarkably stable during the follow-up period (variation ±5%T) (Figure 1 A and B). In 10/53 patients (19%) we observed an increase and in 8/53 (15%) a decrease in JAK2-V617F allelic ratio greater than 5%T. Interestingly, 3/10 patients (1 ET and 2 PV) who showed increase of JAK2-V617F developed secondary myelofibrosis. Twenty six patients (49%) received cytoreductive treatment (hydroxyurea: 24, interferon alpha: 1, anagrelide: 1). Cytoreduction with hydroxyurea did not significantly reduce the JAK2-V617F allelic ratios (Figure 1A) compared to untreated patients (Figure 1B). The only molecular remission was seen in one patient treated with interferon alpha (Figure 1 A). In one patient without cytoreduction JAK2-V617F became undetectable because of transformation to acute myeloid leukemia with blast cells negative for JAK2-V617F. A second patient treated with hydroxyurea showed a pronounced decrease of JAK2-V617F (–47%T in 6 months), but no clinical or laboratory signs of leukemic transformation were present. We conclude that the amount of JAK2-V617F remains very stable in a majority of JAK2-V617F positive patients. Prospective studies will help to elucidate whether increasing JAK2-V617F allelic ratios can predict secondary myelofibrosis or decreasing allelic ratios in absence of cytoreductive therapy (e.g. interferon alpha) are early signs of leukemic transformation. Figure 1A. Figure 1A. Figure 1B. Figure 1B.

Description: Somatic mutations in TET2 occur in patients with myeloproliferative neoplasms and other hematologic malignancies. It has been suggested that TET2 is a tumor suppressor gene and mutations in TET2 precede the acquisition of JAK2-V617F. To examine the order of events, we performed colony assays and genotyped TET2 and JAK2 in individual colonies. In 4 of 8 myeloproliferative neoplasm patients, we found that some colonies with mutated TET2 carried wild-type JAK2, whereas others were JAK2-V617F positive, indicating that TET2 occurred before JAK2-V617F. One of these patients carried a germline TET2 mutation. However, in 2 other patients, we obtained data compatible with the opposite order of events, with JAK2 exon 12 mutation preceding TET2 mutation in one case. Finally, in 2 of 8 patients, the TET2 and JAK2-V617F mutations defined 2 separate clones. The lack of a strict temporal order of occurrence makes it unlikely that mutations in TET2 represent a predisposing event for acquiring mutations in JAK2.

Description: Long term prognosis following allogeneic hematopoetic cell transplantation (HSCT) has greatly improved over the last decades. Therefore, interest of the general health status has become a major topic in the surveillance of long term survivors. To assess health status in very long term survivors (〉10 years follow-up) we conducted a prospective, single center cross-sectional study, evaluating simultaneously 44 recipients and their respective sibling donors. A comprehensive clinical and biological examination was performed. We compared here in a paired analysis the results of routine clinical chemistry tests between recipients and their respective donor (table 1). At the time of this study, recipients and donors had a median age of 44.3 (24–63) and 43.4 (22–61) years respectively, and a median time of 17.5 (11–26) years since HSCT; 22/44 (50%) recipients had chronic graft-versus-host disease (cGVHD). Performance status demonstrated that 81/88 (92%) participants had a Karnofsky Score of 100%, reflecting the overall good clinical condition of both, recipients and donors (p〉0.10). However, clinical chemistry tests of the recipients were systematically abnormal, with increased C-reactive protein (CRP), liver tests, lipids, von Willebrand factor antigen (vWF), creatinine, and decreased albumin, and glomerular filtration rate (GFR), compared with the donors (p

Description: Acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in aplastic anemia (AA), or may selectively involve single lineages, as in pure red cell (PRCA) or in pure white cell aplasias (PWCA). Because of their common cellular immune-mediated pathophysiology, standard treatment for these conditions includes immunosuppression (IS), which may differ according to the specific disease. We investigated an experimental IS regimen based on the anti-CD52 antibody alemtuzumab (MabCampath®, ALE); the study included a phase II/III prospective trial, as well as a collection of retrospective cases. A total of 32 patients have been treated by ALE (18 SAA, 10 PRCA and 4 PWCA), fourteen of them (mostly PRCA) having not received previous IS. The most utilized schedule (as defined in the prospective trial) was 3,10,30,30,30 mg (total dose 103), administered subcutaneously in consecutive days, with adequate premedication; the last dose was amended in PRCA and PWCA patients (total dose 73 mg). In the prospective trial, all patients also received oral low dose cyclosporine A (1 mg/kg) from day 7, and an intensive anti-infectious prophylaxis, which included oral valgancyclovir and cotrimoxazol. All patients completed the treatment with unrelevant injection-related side effect (fever and/or rash in some cases) and absence of laboratory abnormalities. Complete lympho-ablation was observed in all patients within 2–3 days, which persisted for several weeks; transient worsening of neutropenia and/or thrombocytopenia were observed in some cases. The median follow up was 12 months; there were 5 deaths, only one was possibly related to the treatment. In the prospective trial (n=23), infectious events were rare: a single FUO, associated with fatal complication of an underlying atrial fibrillation, other four viral infections (1 VZV with shingles, 2 HSV and 1 flu), all resolving quickly. No CMV or EBV disease was observed, even if 3 border-line CMV reactivations were documented (after discontinuation of the antiviral prophylaxis), promptly resolved by preemptive valganciclovir. One HBV reactivation without hepatitis required lamivudine. The response rate was globally 61% (42% CR and 19% PR), which raised to 73% (50% CR and 23% PR) when only patients with a follow up of at least 4 months were considered. In the more homogeneous cohort of the prospective trial, response rate was analyzed according to the underlying disease. Among 10 AA treated (5 as first line), 7 had an adequate follow up and showed 4 CR (57%) and 2 PR (29%). Response rate was even higher in 10 PRCA (8 as first line): 7 were evaluable for response, with 5 CR (71%) and 1 PR (14%); the 1 non responding patient subsequently showed evolution to MDS. Finally, 2 of 3 PWCA achieved a CR (66%), with the remaining showing early progression to MDS. Among the responding patients, relapses were quite frequent, even while on cyclosporine: 3/6 SAAs, 5/6 PRCAs and 1/2 PWCA. Relapses were successfully treated by additional ALE (as single shoots or complete courses). Immune reconstitution was delayed up to several months, especially affecting the CD4+ compartment; this was also due to additional ALE needed to treat or to prevent relapses. In conclusion, subcutaneous ALE is a feasible and safe IS regimen for patients suffering from immune-mediated marrow failure syndromes. Preliminary results suggest excellent efficacy, even if responses may be quite late (3–4 months); relapses often occur, but can be easily managed by ALE retreatment. ALE is an excellent alternative to standard IS regimen, and deserves systematic investigation in bone marrow failure patients.