Plt Recovery Research Articles

Direct Intra-Bone Cord-Blood Transplantation Overcomes the Problem of Graft Failure Even When Low Numbers of HLA-Mismatched Cord- Blood Cells Are Transplanted, Thus Leading to the Possibility of Use of This Technique in a Large Number of Patients.

Cord blood transplant (CBT) is associated with delayed or failed engraftment in about 20% of patients. We present here the results of intra-bone (IB) transplant of cord blood cells from two Institutions. Fifty-eight consecutive adult patients were included in this study. Patients were: 29 AMLs (10 in first, 3 in second remission and 15 in advanced stage,1 secondary), 15 ALLs (1 in first, 5 in second remission and 9 in advanced stage) 4 CMLs (2 Acc. Phase, 2 Blastic Transformation), 3 refractory Hodgking Disease, 4 refractory Non-Hodgking Lymphomas (HD), 1 Atypical myeloproliferative disease (after HD), 1 SAA and 1 Diskeratosis Congenita (DC). Patients' median age was 36 years (18–66). Human Leukocyte Antigen (HLA) matching was 6/6, 5/6, 4/6 and 3/6 for one, 10, 46 and one patient, respectively. Conditionig Regimen consisted in: fractionated TBI (10–12 Gy)/cyclophosphamide(CTX) (n=42), Fludarabin (Flu)-CTX-TBI 2 Gy (n=6), Treosulfan (Treo)-Thiotepa (TT)-Flu (n=10). Median transplanted cell dose was 2.6 x107/kg (1.4–5.4) and CD34+ cell dose was 0.83 x105/kg (0.42–4.53). Cord blood cells were gently infused in the supero-posterior iliac crest (SPIC) under rapid general anaesthesia. In 21 patients the procedure was performed in both (right and left) SPIC while in 37 patients in either left or right SPIC. No complications were observed during or after the IB infusion of cells. Eight patients with advanced disease died within 12 days from transplant. Two patients did not engraft; one (SAA) had a second IB CBT but engrafted only after mesenchymal stem cell infusion; the other patient (CML-AP) is being re-transplanted. All other patients engrafted. Median time to neutrophils (PMN) and platelet (Plt) recovery was day +23 (14–44) and day +38 (16–64) respectively. Kaplan-Meier probabilities were then 100% for both PMN and Plt. at day +44 and day +64, respectively. Cumulative incidence were 87% (95% CI: 73–97) at day +44 for PMN and 82% (95% CI: 67–95) at day +64 for Plt. At day +30 haematopoietic progenitors Colony Forming Cells frequency were within the limit of normal range both in injected and un-injected site; this shows a rapid recirculation and seeding in the entire hematopoietic system. Chimerism was 100 % donor in all patients from day +60. In 47 patients at risk for acute Graft-versus-Host Disease (GVHD) the score was: grade 0 (n=37) grade I (n=3) and grade II (n=6), grade III (n=1). Seven patients had moderate and two patients extensive chronic GVHD. Causes of death were multiorgan failure(n=8), infections (n=12), PTLD (n=1) and relapse/progression (n=5). Thirty-two patients are alive: 4 in relapse/progression; one with graft failure; and 27 in haematological remission at a median follow-up of 20 months (2–29). Three pediatric patients (age 1– 7 years) were also included in this study: 1 JMML in graft failure after BMT; 1 ALL with t (9;22) in first CR and 1 Hemophagocytic Lymphohistiocytosis (HLH) with active disease following three unrelated BMT. HLA matching was 5/6 (1pt) and 4/6 (2 pts); Conditioning Regimen were: TT+Flu+L-PAM, TBI 12 Gy +CTX+TT, Treo+TT+Flu; median cell dose injected IB was 4.6 x107 TNC (3.0–7.0) and 0.76 x105 CD34+ (0.3–2) respectively. Patients achieved PMN and Plt. recovery at 12-27-47 and 31-34-63 days from IB-injection. All patients are alive, disease-free and full donor chimerism at 249, 172 and 85 days following IB CBT. Our data suggest that direct i.b. cord blood transplant is associated with a very high rate of engraftment even when low numbers of HLA mismatched cord blood cells are transplanted, thus, rendering this transplant possible in a greater number of patients with hematologic diseases. Moreover, high risk pediatric patients may also benefit of IB transplant as salvage procedure.

Induction Intensified Regimens Including Fludarabine or Mylotarg for Acute Myeloid Leukemia Patients: Comparison by Response and Follow-up.

Background. Conventional induction treatment in young Acute Myeloid Leukemia (AML) patients (<= 60 years old) is still represented by the association of antracycline and cytarabine, which offers a complete remission (CR) rate not inferior to 63%. Since many non-randomized trials have recently demonstrated the superiority of intensified regimens, the present gold standard therapy includes the addition of at least a third drug to the classic 3/7 schedule.Aim of the study. We evaluated the safety profile and the efficacy of two four-drugs induction schedules, adding either fludarabine (25 mg/sqm days 1–5) or mylotarg (3 mg/sqm day 6) to idarubicin (6 mg/sqm days 1, 3, 5), cytarabine (1 g/sqm days 1–5), etoposide (100 mg/sqm days 1–5) (FLAIE and MyAIE, respectively).Methods. Sixty-six consecutive AML patients were enrolled either in the FLAIE (N=44, from 2002 to 2005) or in the MyAIE (N=22, from 2005 to April 2007) schedule, with similar clinical and biological characteristics. The median age was 45 and 48 years, respectively. According to kariotype, WBC count and FLT3 status, seventy and sixty-four percent of cases, respectively, were considered at high risk. Consolidation therapy consisted of 2 cycles of ID-AraC and Ida.Results. The complete remission rate was 75% and 59% for FLAIE and MyAIE, respectively (p=n.s.). Death during treatment rates were 5% and 0. After 1 consolidation course the overall CR rate was 80% and 73%. After a similar median follow up, 27 months (1–62) and 21 months (5–42) respectively, 41% of patients are alive in CR in the FLAIE group (12 SCT and 3 ASCT) and 64% in the MyAIE group (7 SCT and 4 ASCT) (p=n.s.; Chi-square, Fisher's exact test). Toxicity was comparable in the two regimens. The median time to ANC recovery (>1.0 x 10^9/L) was 31 and 23 days for FLAIE and MyAIE, respectively. The median time to PLT recovery (>100 x 10^9/L) was 28 and 24 days, respectively. The median time of neutropenic fever episodes for patients was 1 and 1.4 in the 2 groups, respectively. Grade III/IV GI toxicities occurred in 11% and 22% of cases, respectively.Conclusions. These data showed that four-drugs intensified induction therapy is a feasible approach in young AML patients. Recent published trials have demonstrated that fludarabine-based induction chemotherapy in high risk AML patients is able to increase the CR rate offered by conventional treatment. Therefore, the limited number of patients involved in this study, the low administration dosage of idarubicin, and the relevant role of fludarabine in induction therapy, can reasonably justify the lower CR rate obtained in patients treated in the MyAIE schedule, if compared with FLAIE regimen and, above all, with standard chemotherapy. In the light of their efficacy and safety profile, fludarabine and mylotarg, in combination with conventional chemotherapy, represent a promising induction regimen in young AML patients; further analyses and randomized pilot trials will define their definite role.

176: Phase I/II Trial of Bortezomib Maintenance following Autologous Peripheral Blood Progenitor Cell Transplantation as Treatment for Intermediate- and Advanced-Stage Multiple Myeloma

Pts with intermediate- and advanced-stage multiple myeloma were assigned to maintenance therapy with bortezomib beginning a minimum of 3 and a maximum of 4 mos after the day of autologous PBPC transplantation following a standard preparative regimen of high-dose melphalan. Eligibility for the initiation of maintenance required nl organ function with post-transplant neutrophil and plt recovery. Cohorts of 3 pts each were entered at 1 of 3 dose levels of bortezomib (1.0, 1.3, and 1.6 mg/m2) given IV q wk for 3 wks followed by a 1-wk rest period constituting 1 treatment cycle for a total of 8 cycles.

Caspase-3 Activation Is Involved in the Early Megakaryocyte Differentiation, but Not in Platelet Production.

Megakaryocytes (Mgk) differentiate from hematopoietic stem cells through the complex process of endomitosis and cytoplasmic development, and finally form proplatelets in the terminal stage of the differentiation. To elucidate the role of a proapoptotic effecter protein, caspase-3, in megakaryopoiesis and thrombopiesis, we analyzed the phenotype of Mgk in vav-Bcl-2 transgenic (Tg) mice, which overexpress Bcl-2 exclusively in hematopoietic cells by the regulation of vav promotor. As was expected, Bcl-2-overexressing Mgk were remarkably resistant to caspase-3 activation induced by the depletion of thrombopoietin (TPO) from the culture. Percentages of proplatelet formation (PPF) assayed by culturing primary Mgk were slightly increased in the Tg mice. Platelet production from cultured Mgk, which was assayed by flowcytometry, was also slightly increased, indicating normal ability for Plt production from Bcl-2-overexpressing mature Mgk. To further analyze the ability for thrombopoiesis, we assayed the trends of Plt recovery following experimental thrombocytopenia. As Bcl-2-overexpressing mice had giant splenomegaly, we used splenectomized mice, which showed normal Plt counts. After the injection of 5-FU, we observed significant delay of the Plt recovery in the Tg mice, as well as the lack of the overshoot of Plt counts during the recovery phase. To gain insights about the mechanism underlying the delayed Plt recovery, we analyzeded the ploidy of DNA content. In the steady state, peak ploidy of Mgk obtained from the bone marrow of the Tg mice shifted to 2–4N. In a sharp contrast, in Plt recovery phase after 5-FU injection, peak ploidy shifted to 16–32N, and the percentages of Mgk possessing less than 16N ploidy were dramatically decreased. Consistent with this finding, we observed the delay of PPF in Bcl-2-overexpressing cells when Mgk were derived from c-kit+/lineage- progenitor cells in the presence of TPO. These data indicate that differentiation of immature Mgk into mature Plt-producing stage is significantly delayed in the Tg mice, but once the maturation proceeds, Mgk normally produce Plt. Taken together, we conclude that caspase-3 activation is involved in the early stage of the differentiation of Mgk, but not in Plt production.

Phase I/II Trial of Bortezomib Maintenance in Order To Prolong Remission-Duration Following Autologous Peripheral Blood Progenitor Cell Transplantation as Treatment for Intermediate- and Advanced-Stage Multiple Myeloma.

Pts (median age 60 yrs, range 37 – 73 yrs) with intermediate- and advanced-stage multiple myeloma were assigned to maintenance therapy with bortezomib beginning a minimum of three and a maximum of four mos after the day of autologous PBPC transplantation following a standard preparative regimen of high-dose melphalan (200 mg/m2). Eligibility for the initiation of maintenance required nl organ function, post-transplant neutrophil and plt recovery (defined as ANC ≥ 1000 mm−3 and untransfused plt ≥30,000 mm−3). Cohorts of 3 pts each were entered at 1 of 3 dose levels of bortezomib (1.0, 1.3, and 1.6 mg/m2) given IV q wk for 3 wks followed by a 1-wk rest period constituting 1 treatment cycle for a total of 8 cycles. To-date, 29 pts were eligible for study treatment and 23 pts received at least 1 dose of maintenance. Reasons for not proceeding to maintenance included 1 pt with an ANC < 1000 mm−3, 1 pt with a serum creatinine > 2 mg/dl, and 4 additional pts unable to travel to the center. The median t from diagnosis to transplant was 11.4 mos (range, 4.9 to 59.7 mos), and the median t from autologous transplant to the initiation of bortezomib maintenance was 3.7 mos. Protocol adherence was excellent with one pt missing the final dose of treatment and one other pt discontinuing therapy due to personal reasons. After completion of the Phase I portion of the study, the DLT was determined to be 1.6 mg/m2 based on grade 3 diarrhea in 2 pts after 2 and 3 cycles of treatment, respectively. All subsequent pts entered were treated at the MTD of 1.3 mg/m2. Other, non-dose-limiting toxicities included thrombocytopenia (14 pts), neuropathy (12 pts), anemia (12 pts), fatigue (11 pts), neutropenia (8 pts), musculoskeletal pain (7pts), nausea (5 pts), Herpes zoster reactivation (4 pts), diarrhea (3 pts), edema (3 pts), cough (2 pts), as well as upper respiratory infection, chills, fever, dyspepsia, and anorexia (1 pt each). Disease assessment consisted of serum and urine PEP monthly for all pts, and bone marrow aspiration quarterly for those pts with nonsecretory myeloma. Repeat bone surveys were required for pts 3, 9, 15, and 21 mos post-transplant. 2 pts required termination of the study drug, 1 at the end of treatment cycle 8 and the other during cycle 3, due to unacceptable side effects. 2 separate pts terminated the study due to disease progression, 1 during cycle 5 and the other during cycle 6, respectively. One additional pt discontinued further study treatments due to personal reasons during cycle 2. All 23 pts are eligible for response assessment. 13 pts continue to be followed every 3 mos for evidence of disease recurrence in the post-treatment period while 4 others continue on treatment. After a median follow-up from transplantation of 17.1 mos (range 4.1 to 30.3 mo), 6 pts have relapsed while none have died of recurrent myeloma. The current median relapse-free survival is 15.8 mos. This phase I/II trial demonstrates that bortezomib can be safely administered as a maintenance after conventional autologous PBPCl transplantation with an MTD of 1.3mg/m2 given 3 times per month for 8 mos; treatment consolidated remission achieved by autologous transplant as will be presented. These preliminary results, when compared to published results of autologous transplantation for myeloma, suggest that with further follow-up, bortezomib maintenance may favorably impact t- to-recurrence.

A sensitive quantitative single‐platform flow cytometry protocol to measure human platelets in mouse peripheral blood

The NOD/SCID mouse is a widely used model for human cord blood (CB) transplantation. Engraftment is generally estimated with semiquantitative methods, measuring the percentage of human cells among mouse cells. To compare protocols aiming to improve hematopoietic recovery, quantitative methods to enumerate human cells would be preferred. This study describes a single-platform protocol to count human platelets (hPLTs) after transfusion and CB transplantation in the peripheral blood (PB) of the mouse. With an anti-human CD41 antibody against hPLTs and counting beads, the sensitivity to detect hPLTs in mouse blood by flow cytometry was validated. PLT recovery after hPLT transfusions and PLT kinetics after transplantation with CB CD34+ cells was followed in time in NOD/SCID mice. hPLTs could be reliably detected to a level as low as 1 PLT per microL with this single-platform protocol, what appeared to be at least 10 times more sensitive than detection with the dual-platform protocol. To verify the applicability for mouse studies, hPLTs were measured serially in transfusion and transplantation studies in NOD/SCID mice. The results showed that earlier detection of PLT recovery was feasible with the single-platform protocol. A single-platform flow cytometry method can repeatedly measure low numbers of circulating hPLTs in the PB of the same mouse. This method may be helpful in search of new protocols aiming at accelerating PLT recovery after CB transplantation, but also in a number of clinical settings, such as monitoring PLT reconstitution after hematopoietic stem cell transplantation.

Reticulated platelet counts correlate with treatment response in patients with idiopathic thrombocytopenic purpura and help identify the complex causes of thrombocytopenia in patients after allogeneic hematopoietic stem cell transplantation

In thrombocytopenic conditions of unknown origin, quantification of reticulated platelets (RP) in the peripheral blood by flow cytometry has been shown to differentiate increased platelet (Plt) turnover from insufficient Plt production. We used a whole blood flow cytometry method combining thiazole orange and anti-CD41a-staining to assess RP in 71 healthy subjects, six with thrombocytopenic myelodysplastic syndrome (MDS), nine with liver cirrhosis, 14 patients with idiopathic thrombocytopenic purpura (ITP), and 12 patients who had undergone hematopoietic stem cell transplantation (HSCT). Patients with MDS had normal, patients with liver cirrhosis had slightly elevated RP counts compared to healthy subjects. ITP patients had elevated RP counts, and RP >15% were associated with treatment response (P = 0.015). In 7/10 patients after HSCT, an increase of RP preceded Plt recovery, whereas in patients with secondary thrombocytopenia after normal regeneration, the assessment of RP allowed the differentiation between conditions with high Plt turnover, such as GvHD and microangiopathy, indicated by high RP counts, and graft failure, indicated by low RP counts. Our data provide the rationale for prospective studies on the diagnostic and prognostic value of RP counts in larger patient populations with ITP and after HSCT.

Purging "In Vivo" with Alemtuzumab (Campath-1H) before Autologous Stem Cell Transplantation (ASCT) in Chronic Lymphocytic Leukemia (CLL).

Contamination of reinfused stem cells with leukemic cells is a major concern affecting outcomes after ASCT in patients with CLL. In fact, outcomes after ASCT strongly correlate with disease status before transplantation. Persistence of minimal residual disease (MRD), as detected by consensus primer PCR, or the switch from a negative to a positive MRD status during follow-up, are both highly predictive of clinical relapse after ASCT. In order to minimize the risk of contaminating the collection with leukemic cells, in our institution patients who undergo an ASCT receive alemtuzumab before peripheral blood stem cell (PBSC) mobilization, to purge in vivo any residual disease. We evaluated the outcome of ASCT in 20 CLL patients who were pretreated with fludarabine (FAMP) containing regimens and subsequently received alemtuzumab SC (10 mg x 3/w for 6 weeks) to purge MRD. A FAMP containing regimen had been administered as first-line treatment in 18 cases and as second-line in 2 cases; median number of FAMP cycles administered was 6 (range, 4–8). All but 1 PBSC patient was mobilized with Ara-C (800mg/m2/12h x 3 d) followed by G-CSF, while the last patient received only G-CSF. Median age at transplant was 56 y (range, 45–65); all patients (pts)were in CR based on the NCI-WG criteria. Of 20 pts who underwent PBSC harvest, polyclonal IgH rearrangement was evident in 13 pts (65%), as assessed by PCR. The conditioning regimen consisted of 12 Gy TBI plus cyclophosphamide 120 mg/kg in 14 pts < 60 yrs, and Melphalan 180 mg/sqm in 6 pts >60 years. Median number of CD34+ cells reinfused was 17.4 x106/kg (range, 3.1–30.4), and in 13 cases the reinfused product was polyclonal for IgH. The median time for PMN (>500/ml) and PLT (>20000/ml) recovery was 9 (range, 8–11) and 11 (range, 9–13) days respectively. During marrow aplasia 13 patients experienced an episode of fever >38°C with a median duration of 2 days (1–8); in 3 pts the fever was of unknown origin, in 8 cases sepsis was due to Staphilococcus epidermidis, and in 1 case it was due to P. aeruginosa. Intravenous antibiotics were administered in 11 cases, and only 1 patient required intravenous antifungal therapy. One patient died due to a pulmonary fungal infection sustained by Aspergillus Terreus. No incidence of grade 3–4 nonhematologic toxicity was observed. During the 3 months post-transplant 2 pts required hospitalization: 1 for a fever, and the other for acute polyneuropathy. No pathogens were isolated in either case. At 11 months post-transplant 1 patient developed thrombocytopenia. None of the pts developed CMV reactivation, even in the 9 cases, which became CMV positive during alemtuzumab treatment. Herpes Zoster was observed in 2 patients at 5 and 10 months after transplant. At a median of 28 months after receiving alemtuzumab (range, 15–48 months), and a median 17 months after PBSC transplantation (range, 1–41 months), 19 pts are in CR. At the 9- and 12-month post-transplant evaluation, performed on 16 and 12 pts respectively, all but 1 patient showed polyclonal IgH rearrangement. ASCT after sequential treatment with FAMP and Campath-1H is feasible with no significant increase in major infections; a substantial number of patients achieved a sustained polyclonal IgH rearrangement after transplant.

Tandem high-dose chemotherapy (HDCT) and peripheral stem cell rescue (PSCR) in patients (pts) with advanced soft tissue sarcomas (STS), Ewing’s/PNET (ES) and rhabdomyosarcoma (Rhabdo)

9065 Background: To improve progression-free (PFS) and overall survival (OS) a trial of HDCT was conducted in pts with primary high-risk or responding metastatic sarcomas. Methods: Thirteen pts (10 males, 3 females) with ES (7), rhabdo (4), leiomyosarcoma (1) and malignant fibrous histiocytoma (MFH) (1) were accrued from 2/95 through 9/99. Pts initially received induction or salvage chemotherapy with regimens consisting of doxorubicin (D), ifosfamide (I), cyclophosphamide (Cyc), cisplatin (P), vincristine, etoposide, dacarbazine, as well as debulking surgery (9) and radiation therapy (RT) (10). Pts received G-CSF 10μg/kg to procure 14x108 mononuclear cells/kg. Cycle (C) 1 of HDCT consisted of I 14gm/m2 and D 150 mg/m2 administered over 96 hours as an IV infusion, followed by PSCR. C2 consisted of melphalan (M) 75 mg/m2and P 100 mg/m2 on days (d) -11 and -4 followed by PSCR. Results: Eleven of 13 pts completed both cycles of HDCT; 2 pts received C1 only due to reduction in pulmonary function tests and osteomyelitis. No pt died from HDCT. All pts required narcotics for grade 3 mucositis with C1, developed grade 4 neutropenia and required platelet (plt) transfusions. All pts developed neutropenic fever with C1, and 9/11 with C2. The median number of d to neutrophil and plt recovery was 9 (5–18) and 8 (4–31) following C1 and 12 (9–15) and 9 (5–17) following C2. Of the 13 pts enrolled, 2 with advanced ES and 1 with metastatic MFH are alive and free of disease 111, 84 and 60 months post PSCR. The pt with leiomyosarcoma relapsed at 38 months, but was subsequently treated with surgery and RT and is alive at 98 months. The median PFS and OS were 8 and 12 months for pts with rhabdo, and 10 and 22 months for pts with ES. Conclusions: HDCT with D/I followed by M/P is feasible. Although HDCT is unlikely to benefit pts with rhabdo, its role as consolidation therapy in pts with other STS and ES should be assessed in prospective randomized studies. [Supported by NCI Cancer Center Support Grant CA62505] No significant financial relationships to disclose.

Feasiblity of Autologous Stem Cell Transplantation (ASCT) in Chronic Lymphocytic Leukemia (CLL) Patients Treated with Sequential Fludarabine and Campath-1H.

The efficacy of ASCT in pts with CLL may be compromised by the reinfusion of neoplastic cells with the autograft leading to relapse. For this reason in our Institution pts eligible for an ASCT receive Campath-1H before peripheral blood stem cell (PBSC) mobilization to perform a purging in vivo in order to minimise the contamination of the collection. We evaluate the safety and feasibility of APBSCT in fourteen CLL patients pretreated with Fludarabine (FAMP) containing regimens and subsequent Campath-1H subcutaneusly (10 mg x 3/w for 6 weeks). A FAMP containing regimen had been administered as first line treatment in 12 cases and as second line in 2 cases; median number of FAMP cycles administered was 6 (range 4–8). In all but one pts PBSC were mobilized with Ara-C (800mg/m2/12h x 3 d) followed by G-CSF while the last pt received only G-CSF. Median age at transplant was 57.5 y (range 45–63); all pts were in CR, 9 pts showing a polyclonal rearrangement of the IgH at PCR by consensus primer. Conditioning regimen consisted of 12 Gy TBI plus cyclophosphamide 120 mg/kg in 9 pts aged < 60 y, Melphalan 180 mg/sqm in the 5 pts aged > than 60 y. Median number of CD34+ cells reinfused was 17.1 x106/kg (range3.4–30.4), the reinfused product was polyclonal for the IgH in 9 cases. The median time for PMN (> 500/μl) and PLT (> 20000/μl) recovery was 9 (range 8–10) and 11 (range 9–13) days respectively. During marrow aplasia nine pts experienced an episode of fever > 38°C with a median duration of 2 days (1–8); in three pts the cause of fever remained of unknown origin while in the remaining 6 cases a sepsis due to Staphilococcus Aureus was recorded. Intravenous antibiotics were administered in 7 cases; none of the pts required intravenous antifungal therapy. No grade 3–4 extrahematological toxicity and no treatment related deaths were observed. During the 3 months post transplant period two pts required hospitalisation for fever in one case and for acute polineuropathy in the other case; in both cases no pathogens have been isolated. In none of the pts a CMV reactivation was recorded even in the 6 cases showing a reactivation during Campath-1H treatment. An Herpes Zooster infection was observed in 2 pts after 5 and 10 months from transplant. The recovery of CD4+ and CD8+ cells is illustrated in the followig table.CD4 and CD8 reconstitution after ASCTpre ASCTmonth +3month +6month +12 *CD4/ μl579186174CD8/ μl171310377540* 6 ptsAfter a median follow-up of 10 months (3–30) from transplant all pts are in CR. At the three months post-transplant evaluation, performed in 13 cases, all but one pts showed polyclonal IgH rearrangement. ASCT after sequential treatment with FAMP and Campath-1H is feasible with any evidence of increased number of major infections; a substantial number of pts achieved after transplant a polyclonal IgH rearrangement.

A comparison of rhTPO and rhIL-11 following ifosfamide (I), carboplatin (C) and etoposide (E) (ICE) in pediatric patients with recurrent solid tumors or lymphoma

8535 Background: Thrombocytopenia remains the dose limiting toxicity (DLT) for dose intensification of ICE chemotherapy. In an attempt to reduce the incidence of grade IV thrombocytopenia and enhance platelet (plt) reconstitution, we investigated rhTPO (CCG-09717) and rhIL-11 + G-SCF (C9305) after ICE chemotherapy in children with recurrent/refractory solid tumors. Methods: Patients (pts) received I 1800mg/m2 on days (d) 0–4, C 400mg/m2 on d 0–1 and E 100 mg/m2 on d 0–4. In CCG-09717, pts received rhTPO (1.2, 2.4 or 3.6 μg/kg/d) on d +4, +6, +8, +10 and +12. In C9305, rhIL-11 was given at doses of 25, 50, 75, 100 or 125 μg/kg/d beginning d +6. All pts received G-CSF (5 μg/kg/d) on d +6. Results: The median d to ANC ≥1000/mm3 for rhTPO (1.2, 2.4 and 3.6 μg/kg/d) was 20 to 19.5 d, respectively, and rhIL-11, 23 d (25 μg/kg) to 16.5 d (100 μg/kg) for ANC ≥500/mm3. The median d to plt ≥100k/mm3 for rhTPO at 1.2, 2.4 and 3.6 μg/kg/d was 24 to 22 d, respectively (median plt tx d 2 [0–7d]) and for rhIL-11 was 24.5 to 20 d, respectively (median plt tx d 3 [25 μg/kg] to 2 [100 μg/kg]). A dose dependent increase in AUC0-∞ was 2350±630 and 3066±846 ng.hr/mL with mean Cmax (63.3 ± 9.7 and 89.3 ± 15.7 ng/ml) and mean T1/2 (47 ± 13 and 64 ± 42 hr) after the 2.4 and 3.6 μg/kg/d doses of rhTPO, respectively. For rhIL-11, a dose dependent increase in mean Cmax was 7.6–25.5 ng/mL and AUC0-∞ 56.7–208.6 ng.hr/mL at doses of 25–100 μg/kg with mean Tmax 2.4 to 3.1 hr and mean T1/2 4 to 8 hr. There was a significant increase of peripheral blood CFU-megs with rhTPO compared to baseline (0±0 vs 287±97 CFU-meg, p<0.001) vs (0.0 vs 0.0 p=NS) for IL-11. There was no evidence of DLT or antibody formation attributable to rhTPO and no MTD. Papilledema was a DLT of rhIL-11 at doses above 50 μg/kg/d. Periosteal bone changes were seen especially at the 100μg/kg rhIL-11 dose level. 3.7% developed an antibody to rhIL-11. The MTD of rhIL-11 was 50 μg/kg/d. Conclusion: RhTPO vs rhIL-11 in children was associated with a larger T1/2, increased AUC0-∞, less toxicity but similar time to plt recovery and median number of plt tx following ICE. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Wyeth Oncology

Tumor cell depletion of peripheral blood progenitor cells using positive and positive/negative selection in metastatic breast cancer

The clinical relevance of tumor cell purging of hematopoietic progenitor cell grafts has yet to be conclusively determined. Therefore, in addition to the demonstration that a method for graft purification is capable of removing an adequate number of tumor cells, it is critical that the procedure has as benign an impact upon the hematopoietic repopulating potential of the graft as possible. We evaluated tumor cell depletion, recovery of CD34(+) cells and post transplant engraftment kinetics as accepted measures of the effectiveness of an immunomagnetic bead (positive and positive/negative) purging methodology. The patients received either positive selection (CD34 selection alone) or a combination of positive and negative (CD34 selection followed by breast cancer cell depletion) using the Isolex 300 (automated and semiautomated) devices. Immunocytochemistry was used to determine the degree of breast cancer cell contamination before and after the selection procedures to determine the efficacy of the procedure. CD34 enumeration was employed to evaluate the recovery and purity of the CD34-selected cellular products and engraftment indices (days to absolute neutrophil count (ANC) recovery and platelet count (Plt) recovery and transfusion requirements) were evaluated to determine the safety of the procedure. A total of 130 aphereses was performed on 101 patients. Ten pairs of collections were pooled before selection to increase the likelihood of achieving CD34 dose goals after selection. In all, 100 positive selections and 20 positive/negative selections were performed. Of the 10 (10.4%) ICC-positive preselection samples, 2 products showed persistent contamination after processing. The majority of patients (85.4%) required one selection procedure to achieve an adequate CD34(+) selected cell dose. Median CD34(+) cell recovery was > 50% for positive selection procedures and > 60% for the positive/negative procedures. The dose of CD34(+) cells infused ranged from 0.76 x 10(6) CD34(+) cells/kg to 27.7 x 106 CD34(+) cells/kg. There were no significant delays in neutrophil or platelet recovery or infections between any of the treatment groups. CD34 selection alone or in combination with negative selection can result in a significant reduction of contaminating tumor cells in the peripheral blood progenitor cell autograft. Although there was one engraftment failure with the CD34-positive selected cells, transplantation of the selected products after high-dose chemotherapy for metastatic breast cancer did not result in a clinically significant delay in the hematopoietic reconstitutive capacity of the autografts.

Endogenous Interleukin-11 (IL-11) Expression Is Increased and Prophylactic Use of Exogenous IL-11 Enhances Platelet Recovery and Improves Survival During Thrombocytopenia Associated With Experimental Group B Streptococcal Sepsis in Neonatal Rats

ABSTRACT Future preventive and/or concurrent therapy of neonatal sepsis may require the use of adjuvant immunohematopoietic therapy. In the present study, using reverse transcription-polymerase chain reaction, we demonstrated a significant increase in IL-11 mRNA extracted from the femurs of group B streptococcus (GBS)-infected rats during acute thrombocytopenia (platelet count: 65.8 ± 19.3 K/mm 3, n=5) compared to that of uninfected neonatal rats (NR) (635.2 ± 89 K/mm 3, n=5) (174 ± 17% vs. 100%, p<0.001, n=5). We next investigated the prophylactic effect of rhIL-11 on the PLT recovery as well as survival in NR during experimental GBS sepsis. NR received either rhIL-11 (250 μg/kg/d) intraperitoneally for 11 d or sham injections before the induction of experimental GBS sepsis. While experimental GBS sepsis resulted in severe thrombocytopenia in control NR, the rhIL-11 pre-treated group had significantly higher PLT counts (24 hr: 417 ± 50 vs. 221 ± 54 K/mm 3, p<0.01; 36 hr: 276 ± 60 vs. 82 ± 33 K/mm 3, p<0.01; 48 hr: 402 ± 77 vs. 101 ± 82 K/mm 3, p<0.05). Administration of rhIL-11 alone also significantly increased the survival rate at 36 and 48 hrs after GBS inoculation compared to the control group (36 hr: 83% vs. 58%, p<0.05; 48 hr: 50% vs. 18%, p<0.01, n≥30), as did the combination of rhIL-11 with ABS treatment at 36 hrs, compared to the control group (90% vs. 69%, p<0.05, n≥30). These results suggest that endogenous IL-11 gene expression may be upregulated during acute thrombocytopenia and associated bacterial sepsis in NR. This increase in IL-11 gene expression, however, does not appear to prevent severe thrombocytopenia. Furthermore, prophylactic administration of pharmacological doses of rhIL-11 may be potentially beneficial in the management of neonatal GBS sepsis and its associated thrombocytopenia. Future studies are needed to determine the clinical implications of these findings.