Enrica Lerma

In vivo mobilization of karyotypically normal peripheral blood progenitor cells in high-risk MDS, secondary or therapy-related acute myelogenous leukaemia

We have previously reported that mobilization of Philadelphia (Ph) chromosome‐negative progenitors is possible in a significant number of Ph1‐positive acute lymphoblastic leukaemia (ALL) and chronic myelogenous leukaemia (CML) patients. In this pilot study we employed the same approach for patients with RAEB‐t, secondary AML (sAML) and therapy‐related AML (t‐AML). All patients except one had double or complex cytogenetic abnormalities in marrow cells before mobilization therapy. All patients received an idarubicin‐containing regimen (mini‐ICE protocol) followed by rh‐G‐CSF and the first leukapheresis was performed as they were recovering from aplasia. In six out of nine patients the leukapheresis product was entirely karyotypically normal, combined with a significant number of CFU‐GM, CD34+ cells and LTC‐IC. Recovery time from mobilization therapy was short and no patient died as a result of the procedure. To date, three patients have undergone autografting using their karyotypically normal collections, of which two (sAML) are alive with karyotypically normal marrow a few months after autografting.

Mobilization and transplantation of Philadelphia-negative peripheral-blood progenitor cells early in chronic myelogenous leukemia.

PURPOSE Mobilization of Philadelphia (Ph) chromosome-negative progenitors is now possible in many Ph1-positive chronic myelogenous leukemia (CML) patients who had received interferon alfa (IFN-alpha) with no cytogenetic response. In this pilot study, we used this approach in patients without prior IFN-alpha therapy to determine if the number and quality of mobilized progenitors would be increased and to evaluate the potential effect of these cells as autografts. PATIENTS AND METHODS Twenty-two untreated patients were mobilized within 12 months of diagnosis. The treatment regimen consisted of the mini-ICE protocol. Beginning on day +8, granulocyte colony-stimulating factor (G-CSF) was used in all patients. Leukophoresis was performed as the patients were recovering from aplasia, when WBC count exceeded 0.8 x 10(9)/L. RESULTS In 14 patients, (63%) the leukophoresis product was entirely Ph1-negative and in four patients the Ph1-positive cell rate was < or = 7%. Significant numbers of long-term culture-initiating cells (LTC-IC) and CD34+ Thy1+Lin- cells were found in most of the Ph1-negative collections that were tested. Twelve patients underwent autografting with their mobilized peripheral-blood progenitor cells (PBPC) (Ph1-negative collections, 10 patients; major cytogenetic response, two patients). All patients engrafted and are alive; six have Ph1-negative marrow 7 to 15 months after autografting. Posttransplant treatment was IFN-alpha combined with interleukin (IL)-2 because of the recent demonstration of synergistic activity in augmenting cytolytic activity. CONCLUSION Intensive chemotherapy given in early chronic phase of CML is well tolerated and results in high numbers of circulating Ph1-negative precursor cells.

Twelve Years Experience with High-Dose Therapy and Autologous Stem Cell Transplantation for High-Risk Hodgkin's Disease Patients in First Remission After MOPP/ABVD Chemotherapy

High-dose therapy followed by autografting can cure patients with aggressive Hodgkins disease (HD) refractory or with early relapse to first-line combination chemotherapy. On the other hand, the eradication of the disease is rarely achieved in heavily pretreated patients. It has been suggested that patients with HD with very high risk characteristics at diagnosis, often relapse despite appropriate therapy with 7-8 drugs combination. Thus it seems to us that such patients are potential candidates for early autografting during first remission. Twelve years ago, we initiated a pilot study to investigate whether patients with very high risk characteristics, would benefit from early autografting. The application of early autografting was compared with our historical group of patients in complete remission after receiving MOPP/ABVD, who had the same negative prognostic characteristics, refused autografting and who did not receive other treatment after achieving complete remission. Among the 22 consecutive patients entered into the pilot study and autografted, 18 are alive and 17 (77%) remain alive in unmaintained remission at a median of 86 months. One patient (4%) died of interstitial pneumonitis in the transplantation group. Only 8/24 (33%) patients, who did not receive an autograft, are currently alive and disease free at a median of 89 months. In conclusion, the early application of autografting appears to improve the outcome in patients with very high risk HD who achieved remission with MOPP/ABVD.

Evidence of cytogenetic and molecular remission by allogeneic cells after immunosuppressive therapy alone.

An immunosuppressive but not myeloablative regimen followed by HLA‐matched donor mobilized haemopoietic stem cell transplantation was employed in two high‐risk patients. The first patient had refractory anaemia with excess blasts (RAEB) and cytogenetic evidence of translocation 1;3(p36;q21). The second patient had Philadelphia‐negative but p190 BCR‐ABL chimaeric gene positive chronic myelogenous leukaemia in accelerated phase (AP‐CML). The conditioning regimen consisted of fludarabine (30 mg/m2/d, days 1–3) with cyclophosphamide (300 mg/m2/d, days 1–3). Cyclosporine and methotrexate were employed for acute graft‐versus‐host disease (aGVHD) prophylaxis. In both cases the engraftment of donor cells was demonstrated by cytogenetics and short tandem repeat polymorphisms via PCR. Both patients are alive with normal cytogenetic (RAEB) and molecular (AP‐CML) remissions, 100 and 150 d after allografting, respectively. In particular, in the AP‐CML patient, the BCR‐ABL became undetectable and the BCR‐ABL/ABL ratio was <0.0001.

Effective mobilization of Philadelphia-chromosome-negative cells in chronic myelogenous leukaemia patients using a less intensive regimen

To determine if reducing the intensity of the mobilizing chemotherapy protocol used would alter the number and/or quality of the progenitors mobilized in patients with chronic myelogenous leukaemia (CML), we undertook a pilot study. 36 consecutive CML patients previously treated only with hydroxyurea were given mobilization therapy within 12 months of diagnosis. 17 patients were treated by the ICE protocol and 19 patients received the mini‐ICE protocol. The leukapheresis product collected from 22/36 patients (62%) was entirely Ph‐negative. The cytogenetic results between ICE and mini‐ICE‐treated protocols were not significant, although the reduction in median days of hospitalization required for the mini‐ICE versus the ICE protocol was highly significant (P < 0.0001). There was no significant difference in the yield of CD34+ cells and CFU‐GM collected. No patient in the mini‐ICE protocol experienced high‐grade oral mucositis and GI toxicity whereas three such cases occurred with the ICE protocol. No patient died of the mobilization procedure in either group.

Rituximab is effective for extensive steroid-refractory chronic graft-vs.-host-disease.

Chronic graft-vs.-host disease (cGVHD) is the most common cause of late morbidity and mortality after allografting. Patients who fail to respond to steroid-based therapy have poor outcome [1]. There is now mounting evidence implicating B cells in the pathophysiology of cGVHD. Antibodies to Y chromosome-encoded minor histocompatibility antigens are generated after sex-mismatched allogeneic transplantation [2], and the presence of these antibodies has been correlated with the occurrence of cGVHD and a decreased risk of relapse [3]. This finding generates the hypothesis that specific anti-Bcell therapy may be effective therapy for cGVHD. The efficacy of anti-B-cell therapy using the monoclonal anti-CD20 antibody Rituximab for cGVHD has been reported [4 – 8]. Recently, 21 patients with cGVHD were treated by Cutter et al. [9] with 38 cycles of rituximab; the clinical response rate was 70%, including two patients with complete remission. These responses were durable through one year after therapy. Unfortunately, the responses were limited to patients with cutaneous and musculoskeletal manifestations. Here, we describe a patient with extensive liver and gastrointestinal cGVHD combined with severe thrombocytopenia, refractory to cyclosporine, highdose prednisolone and high-dose immunoglobulins who obtained a complete resolution after rituximab. A 28-year-old male presented with bulky mediastinal mass. After a diagnosis of CD20þ diffuse large B-cell non-Hodgkin’s lymphoma was made, the patient received two cycles of conventional chemotherapy (R-CHOP, MACOP-B) with no clinical response. The patient was then treated with highdose therapy (BEAM protocol) and autografting. A positron emission tomography (PET)/computed tomography (CT) scan 2 months after high-dose therapy/autografting showed a 50% decrease in the mediastinal mass. Thirteen months after diagnosis, he received reduced intensity conditioning for allografting fludarabine (30 mg/m for 3 days) and melphalan (70 mg/m for 1 day). Filgrastimmobilized peripheral blood progenitor cells from an HLA-matched sister were harvested and infused into the patient. Prophylaxis against GVHD consisted of cyclosporine and methotrexate. There was prompt hematologic reconstitution after transplantation. On day 28, the patient developed grade III aGVHD with maculopapular rash of the upper torso and diarrhea. Methylprednisolone 10 mg/kg (up to 15 mg/kg) was administered daily. The patient had a good response to steroid therapy in the skin lesions but not in gastrointestinal symptoms. Bone marrow aspirate on day 35 showed full donor chimerism confirmed by polymerase chain reaction of microsatellite markers. On day 100, he developed progressive xerostomia with lichenoid changes of the oral mucosa; subsequently, he experienced complications including coagulase-negative staphylococcal septicemia, cytomagalovirus reactivation, thrombotic trombocytopenic purpura (TTP)-like syndrome, and aspergillus pneumonia. These events were effectively treated with antibiotic, antiviral, plasmaspheresis, and liposomal amphotericin therapies. On day 130,

Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-α) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had 34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P< 0.0001) or bcr-abl/abl ratio (r = 0.86, P < 0.0001) was found. for patients that underwent the procedure in early chronic phase, ph-negative collections showed different levels of bcr-abl expression. bcr-abl transcript numbers varied from a median of 100/μg rna in the first and second leukaphereses, to 500/μg rna in the third and fourth leukaphereses, and 1500/μg rna in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (≤0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (χ2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.

Combined use of autografting and non-myeloablative allografting for the treatment of hematologic malignancies and metastatic breast cancer.

Conventional allografting has relied on a combination of myeloablative and immunosuppressive therapies, which results in substantial morbidity and mortality. To circumvent the problems inherent to the toxicity and treatment related deaths associated with allografting, it has been recently assessed that it is possible to achieve engraftment of donor hematopoietic stem cells (HSC) after immunosuppressive therapy combined or not with myelosuppressive but nonmyeloablative therapy.1-5The basic observation which serves as the rationale for non-myeloablative hematopoietic stem cell transplantation (NST) originates from the documented therapeutic potential of adoptive transfer of alloreactive donor lymphocytes to eradicate resistant malignant host cells escaping maximally tolerated doses of chemoradiotherapy. This is an observation that has provided an option for cure of patients with a large variety of hematologic malignancies,6-8especially chronic myeloid leukemia (CML).7-14

Cytogenetic response to autografting in chronic myelogenous leukemia correlates with the amount of BCR-ABL positive cells in the graft.

OBJECTIVE An important step in successful autografting of patients with chronic myelogenous leukemia is the delivery of a leukemia-free graft. We conducted this study to determine whether the cytogenetic response after autografting was correlated with the number of BCR ABL-positive cells present within the stem cell grafts. MATERIALS AND METHODS By BCR-ABL mRNA quantification, we studied the serial pheresis products from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplantation. We correlated the residual disease within the graft reinfused with the cytogenetic response following transplantation, taking into consideration those responses that lasted 12 months or more. RESULTS Thirty-two patients received a graft with 0-35% Ph-metaphases and 19 received a graft with BCR-ABL/ABL ratio < or =0.01. After a median of 27 months (range, 12-50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months, and 14 of them (78%) received a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.0003-0.01). Twenty-two patients experienced short-lived responses or had >35% Ph-positive cells in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.001-0.01). Therefore, we found a strong association between a BCR-ABL/ABL ratio less than or =0.01 and the achievement of complete or major cytogenetic remission after autografting (chi(2) test, p = 0.0001). Patients reinfused with grafts contaminated at low levels with leukemic cells also showed a longer duration of the response (log-rank test, p = 0.0009). Eleven patients were reinfused with the lowest level of contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients experienced prolonged neutropenia or thrombocytopenia following stem cell reinfusion and nine of them had long-lasting complete or major cytogenetic responses after transplant. CONCLUSION This study demonstrates that the number of BCR-ABL positive cells present in a stem cell graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting. [corrected]

Autografting with Ph-negative progenitors in patients at diagnosis of chronic myeloid leukemia induces a prolonged prevalence of Ph-negative hemopoiesis

OBJECTIVE In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myeloreductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. MATERIALS AND METHODS We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%-100% Ph-negative PBPC for a median time of 28 months (range 18-50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). RESULTS Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%-100% to 0%-25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21-43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term-culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p < 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p < 0.03). CONCLUSION We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.