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Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from allografts to prevent graft versus host disease (GvHD) while conserving useful donor immunity. We developed a semi-closed, GMP-quality, clinical scale SD process where donor-derived lymphocytes are stimulated with patient-derived T-cell antigen presenting cells in an ex vivo mixed lymphocyte reaction (MLR). Alloactivated donor T cells preferentially retain the photosensitizer 4,5-dibromorhodamine 123 (TH9402), rendering them susceptible to elimination by exposure to visible light in a photodepletion device (Kiadis Pharma Inc, The Netherlands). After Food and Drug Administration and Institutional Review Board approval we initiated a clinical trial where HLA-identical sibling recipients with hematological (non T-cell) malignancies received a CD34-cell selected transplant (Miltenyi, Germany) containing less then 1 × 104 T cells/kg together with 5 × 106/kg viable SD donor T cells on day 0, using an age-adapted, radiation-based preparative regimen (FluCyTBI). Low-dose cyclosporine was used as sole immunosuppression in the absence of GvHD. Eleven patients (median age 43 (28–68) years with ALL, MDS, CML, mantle cell lymphoma (MCL), or AML) were transplanted with a median follow-up of 240 (43–400) days. Nine patients were considered high risk. Patients received a stem cell product containing a median of 6.0 (3.9–9.5) ×106/kg CD34+ stem cells in addition to 5×106/kg SD T cells. Absolute lymphocyte recovery was rapid (median 834 (384–2486) cells/μL day 30 post transplant) [Fig A]. Early T cell chimerism was donor-dominated (median 66% (6–95) on day 14, and 97% (82–100) on day 30, and 100% (92–100) on day 45 [Fig B]. One patient received an unmanipulated DLI to treat a delayed fall in T cell chimerism. Three patients developed steroid-sensitive grade II aGvHD of skin (N=2) and gut (N=1) but no grade III–IV aGvHD occurred after transfusion of the photodepleted lymphocytes [Fig C]. Two patients developed limited chronic GvHD. Only one patient, transplanted for refractory MCL, relapsed 340 days after transplant. One patient died of infectious complications and GvHD 330 days after transplant after receiving an unmanipulated DLI in her home country for suspected, but subsequently unconfirmed relapse. Eight patients reactivated CMV but were successfully treated. These results demonstrate for the first time clinical feasibility of photodepletion-based SD stem cell allotransplants in matched siblings. Robust lymphocyte recovery and early donor chimerism with a low relapse incidence in a high-risk population suggest functionality of SD T cells in the absence of severe GvHD, which should allow further reduction of immunosuppression to optimize disease control in future studies.

Reverse seroconversion (RS) known as reactivation of resolved hepatitis B virus (HBV) infection is a complication after allogeneic hematopoietic stem cell transplantation (SCT) in patients exposed to hepatitis B virus. Four hundred thirty six (436) patients with hematological disorder received SCT from an HLA identical sibling in our institute between September 1993 and June 2004. Of these, 103 patients who are 3 or more years post-transplantation have been enrolled in a long-term evaluation protocol. Sixteen had pre-transplant resolved HBV infection (negative HBsAg, positive anti-HBs and anti-HBc antibodies). We investigated the serologic markers of HBV infection in this cohort (median follow-up 59.5 months, range 40–113). Diagnoses included chronic or acute myelogenous leukemia and myelodysplastic syndrome (14), acute lymphoblastic leukemia (1), and severe aplastic anemia (1). Median age at SCT was 32 years (range 10–56). Thirteen patients were positive for anti-HBc and anti-HBs. One had anti-HBc, one had anti-HBs alone, and 1 had no antibodies to hepatitis B. None had detectible HBsAg (HBV surface antigen) at transplant. Twelve patients (median age 39.5 years) received a 12–13.6 Gy total body irradiation (TBI)-based myeloablative SCT followed by a T-cell depleted peripheral blood stem cell transplantation (PBSCT). Four patients (median age 31 years) received a reduced-intensity regimen of fludarabine 125 mg/m2and cyclophosphamide 60 mg/kg, followed by a PBSCT. All received cyclosporine as graft-verse-host disease (GVHD) prophylaxis. Nine developed acute GVHD, and 13 developed chronic GVHD. Nine (56%) patients received immunosuppressive therapy (IST) for chronic GVHD beyond 3 years from SCT. Fifteen (94%) patients were alive with a median follow-up of 59.5 months. One patient with RS of HBV died at 59 months after SCT. Six (38%) patients developed RS of hepatitis B with reappearance of HBsAg, 21–101 (median 29.5) months after SCT. Four (67%) of the 6 developed clinical hepatitis and received Lamivudine treatment with a decrease of HBV viral load. There was no significant difference in median age of 6 patients with, and 10 patients without RS (38 vs 32 years). RS of hepatitis B was associated with prolonged immunosuppressive therapy for cGVHD. Theses results show that RS of HBV post SCT is a significant long-term complication of patients with pre-transplant resolved HBV infection. This emphasizes the importance of long-term hepatitis B serologic monitoring, post-transplant prophylaxis with Lamivudine, HBV vaccine, and prompt initiation of Lamivudine treatment if RS of HBV occurs.

Mutations in telomere repair complex genes TERT (encoding telomerase reverse transcriptase) and TERC (telomerase RNA component) are associated with bone marrow failure, especially acquired aplastic anemia and dyskeratosis congenita. Low telomerase activity leads to short telomeres of leukocytes, predisposing highly proliferative tissues such as the bone marrow to early senescence and exhaustion of the stem cell compartment. Telomere repair gene mutations have been suggested to result in disease anticipation, defined as earlier and/or worsening clinical manifestations in successive generations. We have identified a six-generation pedigree in a large Mennonite family carrying a novel TERT mutation (K570N), which localizes in the catalytic domain with reverse transcriptase activity (RT domain). The index patient is a 26 year old male dairy farmer with a ten-year history of severe aplastic anemia (5% bone marrow cellularity) unresponsive to immunosuppression. A long history of hematologic diseases was well known and named in the family--the patient’s paternal great-great-grandmother had died of a severe blood disorder at age 65 years. However, the great-grandmother and the grandfather had never presented any hematological disease. The patient’s father had myelodysplastic syndrome at age 33 years, evolving to acute myeloid leukemia and death due to failure to recover blood counts after chemotherapy. One of the proband’s paternal aunts had aplastic anemia develop when she was a young woman and has been transfusion-independent for decades in response to chronic androgen therapy. A second proband’s paternal aunt underwent a liver transplant at age 20 for submassive hepatic necrosis with fibrosis. A third proband’s paternal aunt has macrocytosis only at age 47. Two sisters (ages 21 and 23) also have macrocytosis in the absence of other hematological abnormality, and two other sisters are healthy. Genetic analysis showed that TERT K570N mutation is present in the patient’s paternal (including grandfather, three aunts and two affected sisters) but not maternal relatives (making his father an obligatory carrier). The patient’s oldest of three sons, now age four years, has the mutation but is asymptomatic. There was no nail dystrophy, leukoplakia or skin hyperpigmentation in any of the TERT K570N carriers; although the index patient and some of his relatives showed early graying of hair, this characteristic did not track with the mutation. Telomere shortening of leukocytes, as measured by Flow-FISH, tracked to the mutation in three generations analyzed, being shortest in the proband and in his aunt with marrow failure. Mutagenized TERT vectors transfected into telomerase-deficient VA13 cell lines yielded no telomerase activity using the telomeric repeat amplification protocol (TRAP) assay, whereas when wild-type TERT vectors were co-transfected, telomerase activity was approximately half of wild-type transfected only, indicating haploinsufficiency as a mechanism of telomere shortening. Our results confirm the association between aplastic anemia and TERT mutations. The pattern of hematologic disease in this kindred does not support disease anticipation in TERT mutations. Most remarkably, there is a likely relationship between a telomerase gene mutation and hematological malignancy and severe liver disease.

CMV reactivation after allogeneic stem cell transplantation remains a major cause of post-transplant morbidity but may be mitigated by strong memory T cell responses in the transplanted donor T cell repertoire. To induce CMV specific T cell responses in CMV seronegative donors and to boost CMV reactivity in seropositive stem cell donors we vaccinated donors and healthy volunteers with the ALVAC-pp65 live attenuated canarypox vaccine (sanofi pasteur, Lyon, France). A safety study in 4 normal donors confirmed that apart from variable local reactions, an accelerated vaccine schedule was well tolerated. We then studied a similar accelerated regimen giving 1.0 ml intramuscular ALVAC on days 0, 5, and 10 and measured CD4+ and CD8+ T-cell responses to a CMV pp65 peptide library using flow cytometry to measure the frequency of interferon-gamma producing cells. Blood was drawn for testing on days 0, 5, 10, 30, 60, and 90. Positive responses were defined as a two fold increase of interferon-gamma production compared to unstimulated lymphocytes. Four seropositive and eight seronegative individuals were studied. Of the 8 seronegative individuals, 4 were found to have pre-vaccine CD4+ and CD8+ responses to pp65 and were considered to be CMV-experienced individuals. Three of these 4 individuals had a CD4+ and CD8+ response exceeding baseline at some time between days 30 and 90. All 4 CMV-nai¨ve individuals had responses to ALVAC-pp65 by day 30. Of the four seropositive individuals, 3 lacked detectable CD4+ and 2 lacked CD8+ T cell responses at baseline. The 2 subjects that lacked both CD4+ and CD8+ responses at baseline had positive responses with vaccination. To explore the possiblility that immediate early responses might be rapid and transient in CMV-exposed individuals, three donors (one seronegative) were monitored daily during the first 10 days of the vaccine series. All showed significant responses 1–4 days after second vaccination (see figure). Ten patients (8 with hematological malignancies) were recipients of HLA-matched stem cell transplants from vaccinated donors. Four reactivated CMV, two were recipients of T cell depleted transplants and no patient had CMV disease. These results show that ALVAC-pp65 is a well-tolerated vaccine with the potential to induce pp65 cellular immunity in CMV-nai¨ve individuals and boost responses in CMV-experience individuals. However, the kinetics of the CMV response differs according to whether the T cell response exists at baseline but interestingly does not correlate with serostatus. To deliver a high frequency of pp65 specific T cells to transplant recipients, the optimum schedule for CMV exposed donors appears to be after second vaccination. In contrast, longer periods are required in CMV-nai¨ve donors. Based on these findings, we will use appropriately-timed cell collections from ALVAC-pp65 vaccinated donors to explore whether CMV-exposed transplant recipients can be protected from CMV reactivation. Figure Figure

Mutations in telomere repair complex genes TERT(encoding telomerase reverse transcriptase) and TERC(telomerase RNA component) are associated with bone marrow failure, especially acquired aplastic anemia and dyskeratosis congenita. Low telomerase activity leads to short telomeres of leukocytes, predisposing highly proliferative tissues such as the bone marrow to early senescence and exhaustion of the stem cell compartment. Telomere repair gene mutations have been suggested to result in disease anticipation, defined as earlier and/or worsening clinical manifestations in successive generations. We have identified a six-generation pedigree in a large Mennonite family carrying a novel TERTmutation (K570N), which localizes in the catalytic domain with reverse transcriptase activity (RT domain). The index patient is a 26 year old male dairy farmer with a ten-year history of severe aplastic anemia (5% bone marrow cellularity) unresponsive to immunosuppression. A long history of hematologic diseases was well known and named in the family--the patient's paternal great-great-grandmother had died of a severe blood disorder at age 65 years. However, the great-grandmother and the grandfather had never presented any hematological disease. The patient's father had myelodysplastic syndrome at age 33 years, evolving to acute myeloid leukemia and death due to failure to recover blood counts after chemotherapy. One of the proband's paternal aunts had aplastic anemia develop when she was a young woman and has been transfusion-independent for decades in response to chronic androgen therapy. A second proband's paternal aunt underwent a liver transplant at age 20 for submassive hepatic necrosis with fibrosis. A third proband's paternal aunt has macrocytosis only at age 47. Two sisters (ages 21 and 23) also have macrocytosis in the absence of other hematological abnormality, and two other sisters are healthy. Genetic analysis showed that TERT K570N mutation is present in the patient's paternal (including grandfather, three aunts and two affected sisters) but not maternal relatives (making his father an obligatory carrier). The patient's oldest of three sons, now age four years, has the mutation but is asymptomatic. There was no nail dystrophy, leukoplakia or skin hyperpigmentation in any of the TERT K570N carriers; although the index patient and some of his relatives showed early graying of hair, this characteristic did not track with the mutation. Telomere shortening of leukocytes, as measured by Flow-FISH, tracked to the mutation in three generations analyzed, being shortest in the proband and in his aunt with marrow failure. Mutagenized TERTvectors transfected into telomerase-deficient VA13 cell lines yielded no telomerase activity using the telomeric repeat amplification protocol (TRAP) assay, whereas when wild-type TERTvectors were co-transfected, telomerase activity was approximately half of wild-type transfected only, indicating haploinsufficiency as a mechanism of telomere shortening. Our results confirm the association between aplastic anemia and TERTmutations. The pattern of hematologic disease in this kindred does not support disease anticipation in TERTmutations. Most remarkably, there is a likely relationship between a telomerase gene mutation and hematological malignancy and severe liver disease.

The main emphasis of this paper is on fuzzy linguistic hedging, used to modify membership functions. This paper investigates the issues of obtaining new definitions for hedges which exceed the traditional definitions given by Zadeh (and others), particularly seeing that the effect of applying these hedges does not cross beyond the reasonable limits of membership values [0,1] and is still meaningful from the point of view of magnitude of membership value and hence be really effective for an application. Some of the most commonly used hedges are presented, these hedges are very, positively, negatively, slightly more, and slightly less. The effects of these hedges on numeric examples are charted.

The goal of this paper is to present new and different quantifiers for ordered weighted aggregation and illustrate their applicability by a real-life example. The role of these operators in the formulation of multicriteria decision making functions, using the concept of quantifier guided aggregation, is also discussed.

Between 05/1994 and 07/2005, 78 patients with acute leukemia (AML=49; ALL=29) received a T cell depleted stem cell transplant from an HLA-identical sibling, with 1–2 infusions of donor T cells on days 30–100. The conditioning regimen included 12–13.5 Gy total body irradiation (TBI) and cyclophosphamide (Cy) with or without fludarabine. Cyclosporine (CSA) was given as the sole GVHD prophylaxis after transplant. The stem cell source was peripheral blood in 75 and bone marrow in 3. The T cell dose ranged between 0.2 to 2 × 105CD3+ cells/kg. AML and ALL patients were distributed equally among transplant regimen and T cell dose variations. The median CD34 cell dose was 5.4 × 106 cells/kg, (5.4 for AML, and 5.5 for ALL; p=0.50). Actuarial disease free survival (DFS), transplant related mortality (TRM) and relapse rate was 42±8, 20±6 and 40±9 % for AML and 22±10; 35±19 and 64±11% for ALL patients. The median day 30 post-transplantation lymphocyte count (LC30) for AML patients was 418/μl (31–2105) and 778/μl (265–3295) for ALL patients; p=0.063). In multivariate analysis, LC30 emerged as an independent risk factor for transplant outcome in AML but not ALL patients (see figure). In AML patients LC30 above median was associated with superior DFS, lower TRM and lower relapse. CD34 dose and LC30 were highly correlated for AML and ALL analyzed together or separately. Lymphocyte subset analysis of 20 post-transplant samples on day 30 identified a large proportion of CD56+16+ natural killer (NK) cells (median 44%, range 15–86%). Since early post-transplant NK cells are immature and unregulated by KIR-expression they may exert anti-host reactivity. The more favorable outcome for AML but not ALL patients achieving higher lymphocyte counts early post-transplant recapitulates the myeloid leukemia-restricted benefit from superior NK cell recovery observed in AML but not ALL recipients of mismatched T cell depleted stem cell transplants reported by Ruggeri et al (Blood.1999;94:333–9). Figure Figure

Pulmonary function was studied in sixty nine consecutive patients with hematological diseases (65 malignant; 4 benign) 5–12 (median 7.5) years following allogeneic stem cell transplantation from HLA-matched siblings. Fifty-six (81%) patients received total body irradiation-(TBI) based myeloablative bone marrow or peripheral blood stem cell transplantation (MST) for hematological malignancies and 13 (19%) received a fludarabine/cyclophosphamide non-myeloablative peripheral blood stem cell transplantation (NST) for malignant (9) or benign (4) hematological disease. Median age of cohort was 37 years (MST 37, range 10–55; NST 34, range 15–68 years). Thirty nine patients were male. Eleven were smokers. Baseline pulmonary function tests (PFT) showed a median DLCO of 90% predicted (range 59–156%) and FEV1 of 97% predicted (range 64 to132%). Significant pre-transplantation PFT abnormality occurred in 19 (27.5%) patients (DLCO and/or FEV1 <80% of predicted). Twenty five (36%) patients developed acute graft versus host disease (aGVHD) and 43(62%) developed chronic GVHD (cGVHD). At long-term follow-up at a median of 7.5 years after transplant, 32 (42%) patients had PFT abnormalities, 26 with a predominantly restrictive pattern and 6 with a predominantly obstructive pattern. Twenty-two (32%) patients had a mild to moderate decline in PFT (compared with pre-transplant values DLCO/FEV1 value). Of the 32 patients with PFT abnormalities none required supplemental oxygen and only 12 (17.4%) were symptomatic, which included 10 (14.5%) with the most severely depressed pulmonary function (all with predominantly restrictive lung function). In univariate analysis, development of cGVHD (p <0.0001); pre-transplant PFT abnormality (p<0.0001) and a history of smoking (p=0.03); was associated with increased incidence of late PFT abnormality. In multivariate regression analysis, cGVHD (relative risk 36.1; 95% CI 4.1–320) and pre-transplant DLCO/FEV1 of less than 80% of predicted (relative risk 5.5; 95% CI 1.07–29) were independently associated with late decline in PFT. When analyzed for symptomatic PFT abnormality beyond 5 year post-transplant similar results were obtained. The incidence of late PFT abnormality was comparable in both MST (24/56) and NST (5/13) (p=0.51). Our result indicates that a significant proportion of patients developed late PFT abnormality after transplantation, however only a minority are symptomatic. In this series of patients receiving conditioning regimens of different intensity, cGVHD was the only transplant-related risk factor predictive for late pulmonary function defects.