Giovanni Berisso

STROMAL DAMAGE AS CONSEQUENCE OF HIGH-DOSE CHEMO/RADIOTHERAPY IN BONE MARROW TRANSPLANT RECIPIENTS

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

Factors influencing haematological recovery after allogeneic haemopoietic stem cell transplants : graft-versus-host disease, donor type, cytomegalovirus infections and cell dose

Platelet recovery after allogeneic haemopoietic stem cell transplant (HSCT) and predictive factors were analysed in 342 patients with haematological malignancies. All patients were prepared with cyclophosphamide plus total body irradiation, and received an unmanipulated HSCT from an HLA‐identical sibling (n = 270), a matched unrelated donor (n = 67) or an identical twin (n = 5). The source of stem cells was peripheral blood (n = 15) or bone marrow (n = 327). Graft‐vs.‐host disease (GvHD) prophylaxis consisted of cyclosporin A with or without methotrexate. The proportion of patients with < 50 × 109/l platelets on d +50, d +100, d +200 and d +365 after HSCT was 26%, 27%, 14% and 11% respectively. Thrombocytopenia was independent of the degree of complete donor chimaerism. Four variables were predictive of platelet recovery: donor type, acute GvHD, cytomegalovirus (CMV) infection and number of cells infused at transplant. Recipients of an unrelated graft had lower platelet counts (49 × 109/l) on d +50 than identical sibling grafts (108 × 109/l) (P < 0·001) and twin grafts (149 × 109/l) (P < 0·001). Patients with GvHD grades 0, I, II, III and IV had significantly different platelet counts on d +50 (153 × 109/l, 102 × 109/l, 85 × 109/l, 32 × 109/l and 22 × 109/l; P < 0·001) and thereafter. Thrombocytopenia was more frequent in patients with high‐level CMV antigenaemia (> four positive cells/2 × 105) (P < 0·0001) and in patients who received a low cell dose at transplant (≤ 4·1 × 108/kg) (P = 0·009). Platelet counts predicted transplant‐related mortality (TRM) and were higher at all time intervals in patients surviving the transplant. Patients with grade II GvHD and > 50 × 109/l platelets had a lower TRM than patients with grade II GvHD and ≤ 50 × 109/l platelets (14% vs. 40%, P < 0·0001).

Reduced intensity thiotepa-cyclophosphamide conditioning for allogeneic haemopoietic stem cell transplants (HSCT) in patients up to 60 years of age.

Transplant‐related mortality (TRM) remains a major problem in older patients undergoing allogeneic haemopoietic stem cell transplants (HSCTs). We have therefore explored a less intensive conditioning in 33 patients with a median age of 52 years (range 43–60) transplanted from human leucocyte antigen (HLA)‐identical siblings. The underlying disease was chronic myeloid leukaemia (n = 15), acute myeloid leukaemia (n = 6), myelodysplasia (n = 7) or a chronic lymphoproliferative disorder (n = 5); 15 patients (45%) had advanced disease. The regimen consisted of thiotepa (THIO; 10 mg/kg) on day −5 and cyclophosphamide (CY; 50 mg/kg) on days −3 and −2 (total dose 100 mg/kg). The source was bone marrow (BM) (n = 17) or granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood (PB) (n = 16), which were infused without manipulation. Graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and a short course of methotrexate. Mean time to achieve a neutrophil count of 0·5 × 109/l was 17 d (range 11–23) and full donor chimaerism was detected in 79% of patients by day 100. Acute GVHD grade III or IV occurred in 3% of patients. Chronic GVHD was seen in 45% of patients, with a significant difference for PB (69%) compared with BM transplants (23%) (P = 0·009). For BM grafts, the actuarial 2‐year TRM was 6%, the relapse 56% and survival 87%; for PB grafts, these figures were, respectively, 27%, 33% and 68%. Twenty‐five patients are alive at a median follow‐up of 762 d (range 216–1615) and 20 patients (60%) remain free of disease. Thirteen patients (39%) received donor lymphocyte infusion (DLI) either for persisting or relapsing disease and six patients had complete remission. In conclusion: (i) patients up to the age of 60 years can be allografted with reduced intensity conditioning; (ii) the procedure was associated with a low transplant‐related mortality, particularly for bone marrow grafts, because of a lower risk of chronic GVHD; and (iii) DLI were required after transplant in half the patients for persisting disease or relapse.

Foscarnet vs ganciclovir for cytomegalovirus (CMV) antigenemia after allogeneic hemopoietic stem cell transplantation (HSCT): a randomised study

This trial was designed to compare foscarnet with ganciclovir as pre-emptive therapy for CMV infection in patients undergoing allogeneic hemopoietic stem cell transplant (HSCT). Thirty-nine patients were randomized to receive foscarnet 90 mg/kg every 12 h (n = 20) or ganciclovir 5 mg/kg every 12 h (n = 19) for 15 days at the time of development of CMVAg-emia. Primary-end points of the study were (1) outcome of CMVAg-emia; (2) progression to CMV disease; and (3) side-effects of treatment. The secondary end-point was transplant-related mortality (TRM). The two groups were comparable for diagnosis, status of disease, donor type, acute graft-versus-host (aGVHD) prophylaxis, interval between HSCT and CMVAg-emia and number of CMVAg positive cells; the donor and recipient age were borderline older in the foscarnet group. Increments of serum creatinine in the foscarnet group, and cytopenia in the ganciclovir group were controlled by reducing the administered dose: in the first 15 days of therapy 9/20 foscarnet and 10/19 ganciclovir patients had a dose reduction greater than 20% (P = 0.43). Clearance of CMVAg-emia was faster in the foscarnet group although with borderline statistical significance. Failures of treatment occurred in 3/20 patients in foscarnet group vs 8/19 patients in ganciclovir group (P = 0.06): causes of failure were the need for combination therapy to control antigenemia (1/20 vs 5/19), and reactivation during treatment for 2 vs 3 patients, respectively. CMV disease was diagnosed in 1 vs 2 patients (P = 0.5) who subsequently died. The actuarial 1-year TRM was 25 vs 12%, respectively (P = 0.3). This study suggests that foscarnet and ganciclovir are both effective for pre-emptive therapy of CMVAg-emia, although the number of failures would seem to be slightly higher in the ganciclovir patients. Side-effects are seen in both groups and can be managed with appropriate dose reduction.

Adoptive autoimmune hyperthyroidism following allogeneic stem cell transplantation from an HLA-identical sibling with Graves' disease.

Autoimmune diseases which follow allogeneic BMT from a donor who is a patient or a carrier of an autoimmune condition are considered to be a paradigm of adoptive autoimmunity. Seven cases of autoimmune thyroiditis associated with clinical hyperthyroidism have been published to date. In the case reported here a 35-year-old female patient with AML of the M2 subtype received unmanipulated PBSC from her HLA-identical sister who had therapeutically controlled Graves’ disease. Antithyroid antibodies, including thyrotropin receptor (TSHR) antibodies, appeared 1 year after transplant. Clinical hyperthyroidism requiring thyrostatic medication appeared after 2 years. The biological and clinical implications of adoptive, post-transplant autoimmunity are briefly discussed.

Early predictors of transplant-related mortality (TRM) after allogeneic bone marrow transplants (BMT): blood urea nitrogen (BUN) and bilirubin

Transplant-related mortality (TRM) following allo- geneic bone marrow transplantation (BMT) remains a major concern and early identification of patients at risk may be clinically relevant. In this study we describe a predictive score based on bilirubin and blood urea nitrogen (BUN) levels on day +7 after BMT. The patient population consisted of 309 consecutive patients who underwent BMT from sibling (n = 263) or unrelated donors (n = 46) for hematologic disorders between December 1990 and December 1996. Of 27 laboratory tests taken on day +7 after BMT, serum bilirubin (P = 0.02) and BUN (P = 0.007) were found to be independent predictors of TRM in multivariate analysis. The median levels of bilirubin (0.9 mg/dl) and of BUN (21 mg/dl) were then used as a cut-off and a score of 1 was given for values equal/greater than the median. There were 216 patients with scores 0–1 (low risk) on day +7 (bilirubin <0.9 and/or bun <21) and 93 patients with score 2 (high risk) (bilirubin ⩾0.9 and bun ⩾21): the latter had more grade iii–iv acute graft-versus-host disease (P = 0.03), slower neutrophil (P = 0.02) and slower platelet engraftment (P = 0.002). The actuarial 5 year TRM is 22% for low risk vs44% for high risk patients (P = 0.0003). For HLA-identical siblings TRM is 20% vs35% (P = 0.01), for unrelated donors it is 20% vs 65% (P = 0.01). Day +7 score was highly predictive of TRM on multivariate analysis (hazard ratio 1.9, P < 0.01), after adjustment for year of transplant (P < 0.00001), unrelated vs sibling donors (P = 0.001), patient age (P = 0.01) and diagnosis (P = 0.01). These results were validated on an independent group of 82 allogeneic BMT recipients in a pediatric Unit who showed an actuarial TRM of 16% for low risk vs 46% for high risk patients (P = 0.002). This study suggests that it may be possible to identify patients with different risks of TRM on day +7 after BMT: high risk patients could be eligible for programs designed to intensify prophylaxis of post-transplant complications.

Allogeneic bone marrow transplantation (BMT) for adults with acute lymphoblastic leukemia (ALL): predictive role of minimal residual disease monitoring on relapse.

We developed a PCR-based method to monitor clonogenic IgH VDJ rearrangement as a possible predictor of relapse in patients with acute B-ALL after allogeneic bone marrow transplantation (BMT). We studied 23 patients at diagnosis, before and after BMT. At the time of BMT, 13 patients were in first complete remission, eight in second complete remission and two in relapse. Four patients were PCR negative before BMT and remained PCR negative also after BMT (−/− pattern). They are still in remission after a median follow-up of 41 months. Nineteen patients were MRD-positive before BMT: three were PCR negative at first determination after BMT (+/− pattern) and maintain remission. Sixteen patients were PCR-positive at first determination after BMT (+/+ pattern): five became PCR negative (+/+/− pattern) (four with chronic graft-versus-host disease (GVHD) and two after donor lymphocyte infusions (DLI)). Nine patients remained PCR-positive (+/+/+ pattern) (four remain in remission, and six relapsed); two patients died before transplant. In conclusion, PCR negative patients before BMT remained negative post-BMT; many pre-BMT positive patients had initial MRD positivity after BMT: 37% of them achieved a molecular remission with cGVHD or DLI.

Foscarnet prophylaxis of cytomegalovirus infections in patients undergoing allogeneic bone marrow transplantation (BMT): a dose-finding study

This is a dose-finding study using foscarnet for CMV prophylaxis after allogeneic bone marrow transplantation (BMT) in 20 high risk patients (unrelated donors, or T cell depleted, and/or advanced disease). Foscarnet was started on day +1 after BMT and continued until day +100. We explored four different dose levels, patients being entered at the lowest dose level until one patient experiences CMV-reactivation, identified as two consecutive positive CMV antigenemias (CMVAg-emia). The four dose levels expressed as mg/kg/day between days 1 and 30 (induction) and between days 31 and 100 (maintenance) were respectively: dose level I = 60/30 (n = 5); dose level II = 120/60 (n = 4); dose level III = 120/90 (n = 5) and dose level IV = 120/120 (n = 6). All patients showed engraftment: PMN ≥0.5 × 109/l at a median interval of 16, 21, 17, 15 days after BMT, and Plt ⩾30 × 109/l on days 19, 16, 17, 17 respectively. CMVAg-emia was seen in 10 patients at a median interval of 53 days post-BMT (range 33–89) with a median of 10 CMV antigen+ cells (range 1–16). There was a dose effect of foscarnet on CMVAg-emia: respectively 4/5 patients (80%), 2/4 (50%), 3/5 (60%) and 1/6 (18%) at dose levels I, II, III, IV (P = 0.1). CMV disease was seen in 3/9 (33%) at dose levels I, II and 0/11 at dose levels III, IV (P = 0.07). The median number of CMV antigen-positive cells at diagnosis of CMV infection was different: 13 in dose levels I–II and two in dose levels III–IV (P = 0.01). Increased creatininine was seen in 15 patients with a mean of 1.8 mg% (range 1.5–5.7) and was the cause of discontinuation in nine patients (45%). Renal toxicity was reversible in all nine patients. Overall actuarial TRM at 2 years was 31%: 47% for patients at dose levels I–II and 19% for patients at dose levels III–IV. In conclusion, foscarnet exhibits a dose-dependent prophylactic effect on CMVAg-emia, CMV disease and transplant-related mortality with acceptable and reversible renal toxicity. Bone Marrow Transplantation (2000) 26, 23–29.

Alternative donor transplants for patients with advanced hematologic malignancies, conditioned with thiotepa, cyclophosphamide and antithymocyte globulin

Preparative regimens without total body irradiation (TBI) have been reported for alternative donor hemopoietic stem cell transplants (HSCT). Between 7 September 1994 and 7 June 1999 48 patients with advanced hematologic malignancies were conditioned with thiotepa (THIO) 15 mg/kg, cyclophosphamide (CY) 150 mg/kg and antithymocyte globulin (ATG). Donors were HLA mismatched family members (1–2 antigens) (FAM) (n = 24, median age 31 years) or HLA matched unrelated donors (UD) (n = 24, median age 34 years). GVHD prophylaxis was cyclosporine and methotrexate. Stem cell source was peripheral blood (n = 8) or bone marrow (n = 40). Hematologic recovery was seen in 42/46 (91%) evaluable patients and complete chimerism in 31/37 patients (85%). Acute GVHD grades III–IV were seen in 10/46 patients surviving 10 days (21%) and extensive chronic GVHD in 2/36 patients surviving 100 days (5%). Twenty-six patients died (54%), eight of recurrent disease (17%) and 18 of transplant-related complications (37%): main causes of TRM were GVHD (15%), infections (15%) and graft failure (4%). Twenty-two patients (46%) survive with a median follow-up of 877 days (287–1840). The actuarial 3-year survival is 49% for FAM and 42% for UD transplants. Results obtained with this regimen in unrelated grafts for advanced CML (n = 15) were not significantly different when compared to 21 concurrent UD grafts for advanced CML prepared with CY-TBI. In conclusion, the combination of THIO-CY-ATG allows engraftment of alternative donor hemopoietic stem cells. Results are similar when using unrelated matched donors or partially mismatched family donors, and not significantly different when compared to patients conditioned with CY-TBI. Bone Marrow Transplantation (2000) 26, 1305–1311.

Effect of granulocyte colony-stimulating factor on secretion of prolactin, growth hormone, thyroid-stimulating hormone, and cortisol in humans

Abstract In order to assess the endocrine effects of acute and short-term granulocyte colony-stimulating factor (G-CSF), we studied seven patients with myelo- and lymphoproliferative disorders who were given G-CSF to prevent chemotherapy-induced neutropenia. On day 0, the patients received a placebo; on days 1 to 4, they received recombinant human G-CSF (300 μg, subcutaneously). Plasma prolactin, growth hormone, thyroid-stimulating hormone, and cortisol levels were determined at baseline and hourly for 8 hours on days 0 and 1. Additional blood samples were collected at 12 hours on days 0 and 1, and at 9:00 am on days 2 to 5 of the study. After administration of G-CSF, no significant variations in circulating hormone levels were observed with respect to baseline and corresponding placebo levels. The data indicate that short-term G-CSF administration does not cause endocrine abnormalities in patients undergoing this cytokine treatment