Anna Dejana

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.

Cytogenetic analysis in essential thrombocythemia at diagnosis and at transformation: A 12-year study☆

Between 1979 and 1988, 86 patients with clinical and laboratory findings consistent with essential thrombocythemia (ET) were karyotyped at diagnosis. Four patients showed a Philadelphia chromosome and underwent myeloid blastic crisis 2.5-4.5 years later, strongly suggesting a diagnosis of chronic myeloid leukemia. A partial deletion of 13q was seen in another case evolving to leukemia a few months later. Five cases, with normal karyotypes at diagnosis, developed acute transformation after more than 5 years of chronic phase. Four of them showed unusual clonal karyotype abnormalities involving different chromosomal regions. The numerical abnormalities found were trisomy 22 in one case, and trisomy 8 and 19 in another, while structural changes included partial deletion of 5p, partial deletion of 6q, pericentric inversion of chromosome 12, and partial deletion of 20q. These abnormalities have not been previously reported in ET. This investigation confirms the absence of a specific cytogenetic marker for ET, and the infrequent transformation to acute leukemia, often with chromosomal clonal disorders.

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.

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.

Trisomy 8 detection in Ph+ CML patients using conventional cytogenetic and interphase fluorescence in situ hybridization techniques☆

We examined bone marrow (BM) cells from 6 Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) patients in advanced phase of the disease using conventional cytogenetic techniques and fluorescence in situ hybridization (FISH) for detection of an extra chromosome 8. All patients showed mosaicism for trisomy 8 as a secondary chromosome abnormality. For FISH, we used the D8Z5 probe specific for the centromeric region of chromosome 8 and analyzed 300 interphase nuclei and a variable number of mitoses for each patient. The percentages of metaphases carrying trisomy 8 were similar with both techniques, whereas the percentage of interphase nuclei showing three hybridization spots indicative of trisomy 8 was significantly lower than that in metaphases. This finding suggests that cells with a supernumerary chromosome 8 may have a cell cycle time shorter than that of disomic cells.

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]

Late-appearing Philadelphia chromosome in acute lymphoblastic leukemia

We describe the cytogenetic analysis of a patient affected by a common acute lymphoblastic leukemia, L2 type. At diagnosis and first relapse, the karyotype was normal, whereas at the second relapse more than 60% of the examined cells showed a Philadelphia chromosome, without any change in the morphological and immunophenotypical picture. This case confirms the observation that leukemic cells are susceptible to developing a Ph, considered a primary chromosomal abnormality, during the course of the disease.

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.

Autograft Followed by Allograft Without Myeloablative Conditioning Regimen: A New Approach for Resistant Hematologic Neoplasia and Breast Cancer

To decrease the relapse and morbidity risks of allograft in patients with refractory or relapsed hematologic neoplasia and metastatic breast cancer, fourteen patients entered our trial: Hodgkin’s disease (n = 4), non-Hodgkin’s lymphoma (n = 2), advanced chronic myelogenous leukemia (n = 2) (one patient with accelerated phase Ph-negative but p190 BCR-ABL gene positive by RT-PCR and one with Ph-positive blastic phase), metastatic breast cancer (n = 4). Another two patients, with RAEB, received allograft without myeloablative conditioning regimen. After high-dose therapy and autologous stem cell engraftment, the patients were treated with immunosuppressive therapy consisting of fludarabine and cyclophosphamide (Flu-Cy protocol) and then HLA matched donor mobilized stem cells were infused to the patients. GVHD prophylaxis consisted of cyclosporine and methotrexate.