Treatment of metastatic head and neck cancer with mesenchymal stem cells combined with prodrug gene therapy.

This is a clinical observation of a patient treated for metastatic head and neck cancer with mesenchymal stem cells mediated prodrug gene therapy. The cells were applied intravenously. We did not observe any therapeutic effect. However, a temporal bicytopenia was observed.

Tandem autologous stem cell transplantation in multiple myeloma after high-dose chemotherapy with two separate collections: single institution experience.

Presented is a retrospective analysis of multiple myeloma patients transplanted at our institution between November 1993 and August 2007. The objective of this analysis was to assess the feasibility and toxicity of tandem autologous stem cell transplantation (T-ASCT) when stem cells were harvested before first and before second transplantation separately. A total of 90 patients transplanted in our center were analyzed, of whom 43 patients were in tandem transplantation group.The overall response rate (ORR) was 83.7% and 95.1%, estimated five-year overall survival (OS) was 40.1% and 60.0%, probability of five-year event-free survival (EFS) was 18.2% and 25.6%, transplant related mortality (TRM) was 6.3% and 4.6% in the single and tandem transplant group, respectively. In multivariable analysis of all 90 patients, tandem transplantation and ORR attained after induction therapy were favourable prognostic factors for OS (p=0.024 and p=0.002) and EFS (p=0.036 and p=0.008), respectively. In conclusion, tandem transplantation with two separate stem cell harvests, performed separately before the each transplantation, is feasible in majority of patients with acceptable toxicity.

Presence of serum carbonic anhydrase autoantibodies in patients relapsed after autologous stem cell transplantation indicates an improved prognosis.

Here we report patients with Hodgkin's disease and multiple myeloma, who relapsed/progressed after high dose therapy and autologous stem cell transplantation. In patients who developed aplastic anemia type syndrome, spontaneous tumor regression was observed and concomitantly high titers of serum autoantibodies were found. In order to identify the antibody specificity, two-dimensional electrophoresis, blotting and immunoreactions were used to analyze the peripheral blood stem cell extract with autoantibodies containing serum. The unique protein spot visualized exclusively by serum of patients with aplastic anemia type syndrome was identified as carbonic anhydrase I (CA I, accession No. P00915 and Q7M316) by means of mass spectrometry. The specificity of autoantibodies was confirmed by reaction with commercial CAs I, II, IX and XII. Immunoreaction in Western blots with these CA isoforms differed in sera obtained from patients with various types of the disease. Sera of Hodgkin's disease patients reacted with CA I, II and XII; sera of multiple myeloma patients reacted with the CA I, II, XII and IX. Patients developing and/or possessing CA autoantibodies had a significant survival benefit over those who did not develop CA anhydrase autoantibodies. Possible relevance of the presence of CA autoantibodies and clinical outcome is discussed.

Autologous stem cell transplantation in first-line treatment of high-risk aggressive non-Hodgkin's lymphoma.

A single center, retrospective analysis evaluating the outcome of patients with poor-risk aggressive non-Hodgkin's lymphoma (NHL) treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT) as a part of firstline therapy. Forty-seven patients younger than 65 years with diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL) or alk-negative anaplastic large cell lymphoma (ALCL) underwent ASCT between July 1997 and November 2005. Patients with DLBCL and alk-negative ALCL had 2 or 3 age-adjusted International Prognostic Index risk factors. All patients were transplanted after MACOP-B induction therapy followed by 2 courses of DHAP and myeloablative chemotherapy BEM or CBV. The complete response rate to the high-dose therapy was 79% with an estimated 5-year progression-free survival of 66%. At a median follow-up of 35 months (range, 16 to 112 months) the estimated overall survival at five years was 59%. There were 4 treatment-related deaths. Twenty-nine of 47 patients remain in complete remission. Our results confirm the efficacy of high-dose therapy with ASCT during first-line treatment of patients with poor-prognosis aggressive lymphoma, with substantial number of patients cured by using this treatment approach.

Autologous stem cell transplantation with selected CD34+ cells and unmanipulated peripheral blood stem cells in patients with relapsed and refractory Hodgkin's lymphoma: a single centre experience.

With the aim to evaluate the long term outcome after high-dose chemotherapy and autologous stem cell transplantation (HDCT+ASCT) in patients with relapsed or refractory Hodgkin's lymphoma (HL) we performed a retrospective analysis of patients transplanted at our centre. Between January 1993 and December 2005, 126 consecutive patients with relapsed or refractory HL in the age of 16 to 65 years underwent HDCT+ASCT at our centre and were enrolled in this retrospective analysis. Patients were autografted with either CD34+ positively selected or unmanipulated periferal blood stem cells (PBSC). With a median follow up of 69 months (3-162 months), the actuarial 5-y PFS and OS for all patients after HDCT+ASCT were 59% and 72%, respectively. In patients transplanted from 1996 the actuarial 5-y PFS and OS for CD34+ selected group were 64% and 79% and for unmanipulated PBSC group 63% and 66%, respectively. A total of 42/126 (33%) patients died. Treatment related mortality (TRM) was 3% (4 patients). In univariate analysis, chemosensitive disease and increased LDH were the strongest prognostic factors for PFS and OS. Our results confirm the efficacy of HDCT+ASCT in relapsed or refractory HL with acceptable toxicity. The use of CD34+ positively selected stem cells for autografting is feasible, safe and effective procedure.

Establishing the method of chimerism monitoring after allogeneic stem cell transplantation using multiplex polymerase chain reaction amplification of short tandem repeat markers and Amelogenin.

We describe the implementation, optimization, sensitivity determination and first clinical results of polymerase chain reaction (PCR) amplification of polymorphic short tandem repeat (STR) markers and Amelogenin locus coupled with fluorescent detection and capillary electrophoresis in chimerism monitoring of patients transplanted at three different transplant centers using a commercially available multiplex microsatellite assay. The chimerism analysis was performed with genomic DNA extracted from unselected peripheral blood leukocytes of one hundred pediatric and adult patients, who underwent allogeneic stem cell transplantation (SCT) from human leukocyte antigen (HLA) matched or one antigen mismatched related or unrelated donors for malignant (70 patients) and non-malignant (30 patients) diseases. Tested were 79 donor recipient pairs for 15 STR systems and identified an informative marker in all but one of them (98,7%), using 6 selected systems out of these fifteen, that appeared highly informative in our patients population. In 21 sex-mismatched donor recipient pairs we used the Amelogenin locus to distinguish the X and Y chromosome. In sixty-three out of these 100 patients chimerism was regularly analyzed from blood samples taken at various time points after SCT with the median follow up of 17 months. Complete chimerism (CC), maintained over the whole follow-up period, was detected in 24 (38, 1%), stable and decreasing mixed chimerism (MC) in 28 (44, 4%) and increasing MC in 11 patients (17, 5%). Patients with CC, stable and decreasing MC showed a significantly better (p 0,005) overall survival rate (0, 81), compared to those with increasing MC (0, 24). These results demonstrate that STR-based chimerism monitoring with sensitivity above 1% and high informativity (98, 7% of donor recipient pairs) is necessary in establishing the origin of engrafted cells after an allogeneic SCT, in detecting graft rejection and that it may contribute in identifying patients with imminent leukemia relapse.

A significance of additional chromosomal aberrations and other variables on post transplantation outcome of patients with CML.

Chronic myeloic leukemia (CML) is a malignant disease of hematopoietic stem cell characterized by the bcr/abl gene rearrangement. Allogeneic transplantation of stem cells (SCT) is a routinely used treatment method of patients with this diagnosis and remains the only curative mode of treatment. From January 1990 to December 2002, 78 patients with CML underwent allogeneic transplantation and were examined at the Department of Genetics in the National Cancer Institute in Bratislava. Using conventional cytogenetic and FISH 6 patients (7.7%) showed additional chromosomal changes before SCT. These patients had statistically worse post transplantation prognosis compared to the patients without additional changes before SCT (mean survival in month+/-standard error (58.08 (+/-6.70) vs. 5.17 (+/-0.98), p-value=0.001), patient mortality (67% vs. 31%)). In addition five other variables were evaluated for transplant outcome, namely, patient's age at the time of transplantation, sibling or non-sibling donor, higher than 1st chronic phase CML, time from diagnosis to transplantation and sex of donor and recipient. Only the comparison of HLA-identical sibling transplantation to unrelated donor transplantation was statistically significant (mean survival in month- 56.6 (+/-7.2) vs. 13 (+/-0.0), patient mortality 31% vs. 67%).

Use of selected CD34+ cells in the treatment of relapsed/progressive HD: experiences from a single center.

BACKGROUND: Treatment of HD using positively selected stem cells can achieve a durable CR in almost 80% patients, with a median follow-up of 24 months. We have found the use of positive selected CD34+ cells in the treatment of relapsed/progressive HD after high-dose chemotherapy to be a safe procedure with promising results. METHODS: Positively selected (CD34+) stem cells were employed for autografting in the patients with HD after high-dose chemotherapy. RESULTS: Between April 1996 and February 2001, 28 patients with relapsed/progressive HD were autografted with positively selected CD34+ cells at our Institute. All patients are alive and we did not observe any deaths as a result of toxicity. From this group, 22 (78.6%) patients are in durable CR, with a median follow-up of 24 months (range 7-65 months). Six (21.4%) patients relapsed but are now in CR after radiotherapy and mini-allogeneic transplantation (one patient) or radiotherapy (five patients). DISCUSSION: Autologous transplantation with positively selected CD34+ cells in relapsed HD is a safe and effective procedure in the treatment of this disease.

Sequential intermediate high-dose therapy with etoposide, ifosfamide and cisplatin for patients with germ cell tumors.

Intermediate high dose VIP (etoposide, ifosfamide, cisplatin) achieved comparable efficacy and improved tolerance in comparison with high-dose chemotherapy plus PBSC in poor risk germ cell tumors. The aim of this study was to confirm the effectivity and tolerance of this regimen in clinical practice. Twenty-five consecutive patients, 9 previously untreated with poor prognosis and 16 relapsed, were treated with 1.6 VIP or 1.9 VIP+PBSC. A relative dose intensity of 1.6 VIP was used in 14 patients and 11 patients received the intensity of 1.9 VIP. Clinical response was achieved in 56% of patients. Fifty-eight percent of patients have survived more than 1 year and 44% more than 2 years. No significant difference was noted between previously treated and untreated patients, as well as between the patients on 1.6 VIP and 1.9 VIP, with the exception of improved 1-year survival of patients on 1.9 VIP. One of four cisplatin-refractory patients achieved durable partial remission with a normal level of tumor markers. Serious non-hematological toxicity was rare. Myelotoxicity of 1.9 VIP was less serious in comparison with 1.6 VIP regimen, but the difference was not significant. Sequential intermediate high-dose therapy is an effective and tolerable regimen for patients with poor risk germ cell tumor as well as for relapsed patients.

ADP is a substrate for the AAUAAA-directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts.

The specific poly(A) addition reaction catalyzed by crude nuclear extracts from HeLa cells can use ADP as efficiently as ATP as the donor of AMP residues. Both the ADP- and ATP-supported reactions require an intact upstream polyadenylation signal sequence element (AAUAAA). The mutated signal sequence (AACAAA) supports neither reaction. The ADP-supported poly(A) addition reaction can be resolved by glycerol gradient centrifugation of the crude nuclear extract into two components which are active when recombined but are inactive individually. The ATP-supported poly(A) addition is reconstituted by recombining the same gradient fractions, but the activity is lower than that supported by ADP, suggesting that an ATP-specific factor has been removed. A 150 mM KCl fraction DEAE-Sepharose of the nuclear extract, also devoid of the ATP-supported poly(A) addition reaction, retains a normal ADP-supported reaction. Together, these data show that ADP is a substrate for polyadenylation, and suggest that different factors might be required to induce ADP- or ATP-specificity in the poly(A) addition reaction.

In vitro transport of F1-ATPase beta-subunit into mitochondria of Zajdela hepatoma and rat liver.

Transport of precursor of F1-ATPase beta-subunit into isolated mitochondria of Zajdela hepatoma and rat liver was examined. The hepatoma mitochondria were more active in the process than the liver organelles indicating that the relative F1-ATPase deficiency in the tumor mitochondria does not result from an impaired transport of F1-ATPase subunits into the tumor organelles. Similar results were obtained using digitonin-treated rat hepatocytes and Zajdela hepatoma cells instead of isolated mitochondria. The suitability of the digitonin-treated cells in the study of protein transport into mitochondria in vitro is demonstrated and the advantages of this system over isolated mitochondria are discussed.

Mitochondrial ATPase of Zajdela hepatoma. II. Mitochondria of Zajdela hepatoma contain less adenosine triphosphatase than mitochondria of rat liver.

The specific activity and the content of ATPase in mitochondria of rat liver and Zajdela hepatoma were compared. The specific activity of ATPase in sonicated mitochondria and in mitochondrial membrane fraction of rat liver was almost two times higher than the specific activity in the corresponding fraction of Zajdela hepatoma. Accordingly, the autovertin binding capacity of rat liver mitochondrial membrane fraction as well as the yield of F1-ATPase from this fraction were about two times higher than those of the mitochondrial membrane fraction of Zajdela hepatoma. The results show that mitochondria of Zajdela Hapatoma possess about half amount of ATPase present in rat liver mitochondria.