What Causes Aplastic Anaemia: Results of Transplants from Genetically-Identical Twins.

Background: Aplastic anaemia has diverse aetiologies. Distinguishing between these is, in part, testable by analyzing results of haematopoietic cells transplants between genetically-identical twins one of whom has aplastic anaemia. Objective: Describe outcomes of genetically-identical twin transplants for aplastic anaemia with and without pretransplant conditioning. Methods: We interrogated data from an observational database of 59 consecutive recipients of genetically-identical twin transplants for aplastic anaemia reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) 2000-2019. Results: 38 subjects were male. Median age was 18 years (Interquartile Range [IQR], 11-32 years). Median interval from diagnosis to transplant was 2 months (IQR 1-3 months). 11 subjects received a 1st transplant without pretransplant conditioning. 2 of recovered normal bone marrow function. The other 9 received a 2nd transplant with pretransplant conditioning 7 of whom recovered. 48 subjects received pretransplant conditioning before a 1st or 2nd transplant all of whom recovered. Conclusion: Only some genetically-identical twins with aplastic anaemia recover normal bone marrow function after a 1st haematopoietic cell transplant without pretransplant conditioning whereas most subjects recover when a transplant is preceded by pretransplant conditioning. These data are consistent with an immune-mediated aetiology of aplastic anaemia in most cases.

Circulating cytokeratin 18 fragment m65-a potential marker of malignancy in colorectal cancer patients.

Soluble cytokeratin 18 fragments (M30, M65) are released from human cancer cells during cell death and hold potential as biomarkers in colorectal cancer characterized by frequent metastatic spread. A total of 62 colorectal cancer and 27 control patients were included in the study. M65 (necrosis and apoptosis) and M30 (apoptosis) were quantified preoperatively (n = 62) and postoperatively (n = 31) using specific enzyme-linked immunosorbent assays. Presence of disseminated tumor cells (DTC) in the bone marrow was assessed by staining of A45-B/B3-positive cells in aspirates. M65 was significantly elevated in patients with International Union against Cancer stage I and IIA tumors compared to controls. A subgroup (19/31) exhibited a significant (p < 0.05) decrease of M65 after tumor surgery (503.9 +/- 230.7 to 342.6 + 94.8 U/l; -32.0 +/- 16.5%), in contrast to 12 patients who revealed higher M65 levels postoperatively (386.5 +/- 128.5 to 519.1 +/- 151 U/l; +37.4 +/- 32.3%). DTC in bone marrow were found in 10% (2/19) of patients with decreasing and 50% (6/12) of the patients with increasing M65 serum concentrations after surgery (p = 0.028). In conclusion, M65 as marker is likely to be valuable to identify patients with a high incidence of systemic disease.

Risk factors affecting outcome of second HLA-matched sibling donor transplantations for graft failure in severe acquired aplastic anemia.

We examined transplantation outcomes after a second HLA-matched sibling transplantation for primary (16%) or secondary (84%) graft failure in 166 patients with severe acquired aplastic anemia (AA). Two-thirds of these patients has a performance score < 90. In most cases (88%), the same donor was used for both transplants, for both transplantations, and 84% of the second transplantations used bone marrow grafts. We identified 2 prognostic factors: intertransplantation interval (surrogate for primary graft failure and early secondary graft failure) and performance status. Shorter intertransplantation interval ( 3 months from first transplantation was 56% and 76%, respectively. The corresponding probabilities in patients with lower performance scores were 33% and 61%. The predominant cause of failure after second transplantation was nonengraftment (in 72 of 166 patients), most commonly in patients with primary or early secondary graft failure (51 of 72; 71%). Our data indicate that novel approaches, including conditioning regimens with greater immunosuppression, should be explored for these patients.

Immunomagnetic CD45 depletion does not improve cytokeratin 20 RT-PCR in colorectal cancer.

BACKGROUND: Cytokeratin 20 reverse transcriptase polymerase chain reaction (CK20 RT-PCR) of blood and bone marrow specimens has been suggested for assessment of hematogenously disseminated tumor cell (DTC) spread in colorectal cancer (CRC) patients. Considerable discrepancies among the studies reported indicate a need for better evaluation procedures. We investigated whether mononucleated cell (MNC) enrichment by Ficoll density gradient centrifugation followed by immunomagnetic depletion of CD45-positive cells (extended enrichment) allows better detection of DTC-associated CK20 mRNA compared to MNC enrichment by Ficoll density gradient centrifugation alone (Ficoll enrichment). METHODS: We analyzed 53 samples [38 peripheral blood (PB), 15 bone marrow (BM)] from 38 CRC patients. Extended enrichment was performed for 30 specimens (PB and BM, n=15 each), and Ficoll enrichment for 23 blood specimens. Total RNA was extracted, reverse-transcribed and analyzed by real-time RT-PCR using a LightCycler instrument. RESULTS: Despite extended enrichment, 10 PB and 8 BM samples could not be analyzed because of low cellular yield. The depletion efficiency of CD45 separation was 2 log. RT-PCR of the housekeeping gene PBGD resulted in high and varied crossing point values (mean 37.1+3.0) for five PB and seven BM specimens. Ficoll enrichment yielded 23 analyzable blood specimens for which the mean crossing point value was 26.7+0.5 in PBGD RT-PCR. CK20 RT-PCR of 23 blood samples (all from Dukes D patients) revealed CK20 transcripts in four cases (17%). CONCLUSIONS: Extended enrichment was not superior to Ficoll enrichment; in fact, the sensitivity was lower. Improvement of the reported CK20 RT-PCR assay of Ficoll-enriched MNC populations is warranted.

Long-term results of autologous stem cell transplantation for Hodgkin's disease (HD) and low-/intermediate-grade B non-Hodgkin's lymphoma (NHL): a report from the Austrian Stem Cell Transplantation Registry (ASCTR).

Between 1990 and 2001, 68 patients with advanced Hodgkin's disease (HD) and 86 patients classified as low-/intermediate-grade B non-Hodgkin's lymphoma (NHL) were reported to the Austrian Stem Cell Transplantation Registry (ASCTR). Following autologous stem cell transplantation (SCT) for HD, overall survival was 56% [95% confidence interval (CI): 40-72%] with a disease-/progression-free survival of 49%, reaching a plateau at 5 years. Using multivariate Cox regression analysis BEAM conditioning (carmustine, cytarabine, etoposide and melphalan) was predictive for favourable outcome, better disease-/progression-free survival and a significantly lower risk for relapse. The cumulative incidence of relapse was 30%, even for patients in complete remission at time of SCT. The cumulative risk for developing a secondary malignancy increased continuously over time, achieving 20% at 7 years and 46% at 10 years with previous radiotherapy as the only risk factor in the multivariate analysis. Overall survival for NHL patients was 45% (95% CI: 26-64%) with a disease-/progression-free survival of 26% at 7 years. In the multivariate Cox regression analysis stage of disease at time of SCT was the most powerful parameter for overall survival, disease-/progression-free survival and relapse. Mantle cell lymphoma, greater than or equal to three lines of previous therapy, and a conditioning regimen other than BEAM were also predictive for death. The main reason for treatment failure was relapse (cumulative incidence 54-75%). Because of the high risk of relapse/progression in both disease categories and the additional high rate of second malignancies in HD patients, allogeneic stem cells should be considered a valuable alternative for selected patients. The efficacy of allotransplantation following reduced-intensity conditioning should be tested in randomised trials.

A rare case of interstitial pneumonitis after tandem high-dose melphalan conditioning and autologous stem cell transplantation in multiple myeloma.

A 57-yr-old woman with multiple myeloma received an autologous tandem transplant at a 4-month interval. She was conditioned twice with 225 mg/m2 melphalan. After the second transplant, interstitial pneumonitis (IP) ensued. The clinical course was life threatening and mechanical ventilation was required for 32 d. All attempts to identify an infectious agent failed. A presumptive diagnosis of idiopathic IP, possibly related to melphalan toxicity, was made. High-dose methylprednisolone administration led to rapid and durable improvement. Melphalan was employed for conditioning in the tandem setting with an interval of only 3-4 months between two courses or a dose elevation to 225 instead of 200 mg/m2, may have induced IP which responded favorably to methylprednisolone.

Diagnostic and prognostic value of colony formation of hematopoietic progenitor cells in myeloid malignancies.

Hematopoietic progenitor cells are capable of forming colonies of mature blood cells in semisolid media in response to specific growth factors. Colony assays have been extensively used for many years to study normal and malignant hematopoiesis in vitro. In fact, these assays have provided an excellent research tool for investigating growth and differentiation of progenitor cells in response to positive and negative regulators of hematopoiesis. However, apart from their role in basic research, colony assays are also widely used in routine clinical practice in the diagnosis of various hematologic disorders, such as aplastic anemia, myelodysplastic syndromes and myeloproliferative disorders. This review summarizes our current knowledge on the diagnostic value and prognostic significance of the growth of progenitor cells in peripheral blood and bone marrow in patients with myeloid malignancies.

Unusual karyotype aberrations involving 2p12, 3q27, 18q21, 8q24, and 14q32 in a patient with non-Hodgkin lymphoma/acute lymphoblastic leukemia.

The t(2;18)(p12;q21), known as a rare variant of the t(14;18)(q32;q21), together with t(3;14)(q27;q32), t(8;15)(q24;q22) and two other unusual translocations involving chromosomes 6, 9, 12, and 13, were demonstrated in the bone marrow cells of a 70-year-old male with suspected non-Hodgkin lymphoma/acute lymphoblastic leukemia. The complex chromosomal aberrations were identified by chromosome banding analysis and by fluorescence in situ hybridization (FISH) with whole chromosome painting probes, centromere-specific alpha-satellite probes, and probes specific for genomic sequences of some likely to be involved candidate genes. Several but not all of the chromosomal aberrations could be proved by multicolor FISH. Possible mechanisms leading to this unusual karyotype commonly associated with different histologic lymphoma subtypes and their prognostic implications are discussed.

Interleukin-10 inhibits in vitro hematopoietic suppression and production of interferon-gamma and tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with aplastic anemia.

INTRODUCTION: Mononuclear cells (MNC) from patients with aplastic anemia (AA) can inhibit hematopoietic colony formation from normal bone marrow (BM) cells. Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are considered as soluble mediators of BM suppression in AA. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate the hematopoietic effects of MNC from patients with AA. METHODS: Using coculture experiments we studied the effect of recombinant human IL-10 (rhIL-10) on the in vitro hematopoietic suppression by peripheral blood (PB) MNC from AA patients. RESULTS: PBMNC (5 x 10(5)/ml) from seven patients with AA caused a 40-100% reduction of normal burst-forming unit-erythroid (BFU-E) growth and a 38-91% reduction of colony-forming unit-granulocyte/macrophage (CFU-GM) growth, respectively, in semisolid cocultures. IL-10 was highly effective in reversing growth inhibition in these cocultures. Addition of 10 ng/ml IL-10 to cocultures significantly restored growth of BFU-E in all seven cases and growth of CFU-GM in five of seven cases, respectively. The effect was dose dependent and correlated with decreased IFN-gamma and TNF-alpha production in suspension cultures. Using intracellular cytokine staining it was found that increased TNF-alpha production in AA cells was derived from both CD4+ and CD8+ cells, whereas aberrant IFN-gamma synthesis was only detected in CD8+ cells. CONCLUSION: IL-10 is effective in reversing in vitro hematopoietic suppression by PBMNC from AA patients. These results suggest therapeutic evaluation of rhIL-10 in patients with AA.

Factors affecting long-term outcome after allogeneic haematopoietic stem cell transplantation for acute myelogenous leukaemia: a retrospective study of 172 adult patients reported to the Austrian Stem Cell Transplantation Registry.

Between 1982 and 2000, 172 patients with acute myelogenous leukaemia (AML) received haematopoietic stem cell transplants (SCT) from related (n = 132) or unrelated (n = 40) donors at four Austrian transplant centres and their results were reported to the Austrian Stem Cell Transplantation Registry. Conditioning for SCT consisted of cyclophosphamide and total body irradiation in 156 (91%) patients. Graft-versus-host disease (GVHD) prophylaxis was with standard cyclosporine and methotrexate in 95 (55%) patients. Median post-transplant follow-up was 5.6 years (range, 0.2--16.7). Multivariate analysis of transplant-related mortality (TRM) identified four variables associated with a lower risk: disease status of first complete remission (CR) at SCT, patient age of 45 years and younger, transplant performed during or after 1995, and lack of acute GVHD. Variables associated with significantly improved leukaemia-free survival were: bone marrow as the stem cell source, disease status of first CR at SCT, and occurrence of chronic GVHD. In multivariate analysis, transplantation performed during or after 1995, first CR at SCT, occurrence of limited chronic GVHD and lack of acute GVHD grades III to IV were associated with increased overall survival. Based on these analyses, options for the improvement of results obtained with allogeneic SCT in patients with AML could be defined.