Regina Kunzmann

New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients

We have generated a large, unique database that includes morphologic, clinical, cytogenetic, and follow-up data from 2124 patients with myelodysplastic syndromes (MDSs) at 4 institutions in Austria and 4 in Germany. Cytogenetic analyses were successfully performed in 2072 (97.6%) patients, revealing clonal abnormalities in 1084 (52.3%) patients. Numeric and structural chromosomal abnormalities were documented for each patient and subdivided further according to the number of additional abnormalities. Thus, 684 different cytogenetic categories were identified. The impact of the karyotype on the natural course of the disease was studied in 1286 patients treated with supportive care only. Median survival was 53.4 months for patients with normal karyotypes (n = 612) and 8.7 months for those with complex anomalies (n = 166). A total of 13 rare abnormalities were identified with good (+1/+1q, t(1q), t(7q), del(9q), del(12p), chromosome 15 anomalies, t(17q), monosomy 21, trisomy 21, and -X), intermediate (del(11q), chromosome 19 anomalies), or poor (t(5q)) prognostic impact, respectively. The prognostic relevance of additional abnormalities varied considerably depending on the chromosomes affected. For all World Health Organization (WHO) and French-American-British (FAB) classification system subtypes, the karyotype provided additional prognostic information. Our analyses offer new insights into the prognostic significance of rare chromosomal abnormalities and specific karyotypic combinations in MDS.

Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2 '-deoxycytidine

Decitabine (5‐aza‐2′‐deoxycytidine) acts as a powerful demethylating agent in vitro. Clinically, low‐dose decitabine ameliorates cytopenias including induction of trilineage responses in ≈50% of patients with high‐risk myelodysplastic syndrome (MDS). We examined the incidence and kinetics of cytogenetic responses to decitabine in these patients. Of 115 successfully karyotyped patients, 61 (53%) had clonal chromosomal abnormalities prior to treatment. Major cytogenetic responses were observed in 19 patients (31% of those with abnormal cytogenetics, 17% of all patients by intention‐to‐treat) after a median of three courses (range, 2–6) until best cytogenetic response. Progressive decrease of the abnormal clone over time was also determined using fluorescence in situ hybridization (FISH) analysis in two patients. Median duration of cytogenetic responses was 7·5 months (range, 3–15). Analysis of response by the International Prognostic Scoring System (IPSS) cytogenetic risk groups revealed three out of five cytogenetic responses (60%) in the IPSS ‘low‐risk’ group, 6 out of 30 with ‘intermediate risk’ (20%) and 10 out of 26 in the ‘high‐risk’ group (38%). Median survival in these cytogenetic subgroups was 30, 8 and 13 months respectively. The relative risk of death in patients achieving a major cytogenetic response was 0·38 (95% confidence interval 0·17–0·88) compared with patients in whom the cytogenetically abnormal clone persisted (P = 0·0213). In conclusion, repeated courses of low‐dose decitabine induce cytogenetic remissions in a substantial number of elderly MDS patients with pre‐existing chromosomal abnormalites; these are associated with improved survival compared with patients in whom the cytogenetically abnormal clone persists. Patients with ‘high‐risk’ chromosomal abnormalities may particularly benefit from this treatment.

Adoptive immunotherapy for relapsed multiple myeloma after allogeneic bone marrow transplantation (BMT): Evidence for a graft-versus-myeloma effect

Adoptive immunotherapy with donor-derived buffy coat cells for relapsed hematological malignancies after allogeneic BMT is an established and highly effective treatment. We report a patient who relapsed on day +330 after allogeneic sibling BMT for multiple myeloma with multiple solid subcutaneous tumors consisting of plasma cells. Histology and immunocytology of the bone marrow did not show plasma cell infiltration. After cessation of the immunosuppression consisting of cyclosporine and methylprednisolone, a total of 6.2 × 107/kg recipient body weight CD3+ T cells derived from the donor by leukapheresis were transfused on 4 consecutive days. To enhance the T cell effect six doses of 5 million units alpha interferon were given subcutaneously. Five days later the tumors started to shrink and have completely vanished since day ×400 after BMT. The patient developed acute GVHD grade III of the liver and gut which was treated by reinduction of various immunosuppressive drugs. Up to now there is no evidence for relapse of the multiple myeloma, but the patient suffers from extensive chronic GVHD (gut and liver). This is the first report to demonstrate a graft-versus-myeloma effect for relapse with solid tumor manifestation after sibling BMT with donor-derived buffy coat cells as adoptive immunotherapy.

Allogeneic bone marrow transplantation from unrelated donors using in vivo anti‐T‐cell globulin

Despite improvements in HLA typing, graft‐versus‐host disease (GVHD) continues to impair the results after volunteer unrelated donor bone marrow transplantation (VUD‐BMT) in adult patients compared with matched sibling BMT. Here, the outcome after VUD‐BMT using a specific regimen with high‐dose anti‐T‐lymphocyte globulin (ATG) was analysed. Fifty‐five adult patients, median age 34 years (range 17–55 years), with acute or chronic leukaemia or myelodysplastic syndrome (MDS) were transplanted in first complete remission (CR1)/first chronic phase (CP1) (early disease) (n = 21) or in advanced (CR2/CP2, no remission) disease (n = 34) from an unrelated marrow donor. GVHD prophylaxis consisted of ATG‐S (Fresenius) 60–90 mg/kg b.w. prior to transplantation, in addition to cyclosporin A and short‐course methotrexate. Graft failure did not occur and white blood cell count (WBC) > 1·0 × 109/l was reached at median day +16. The cumulative incidence of acute (a)GVHD grade II–IV was 15% [95% CI (8%, 28%)] and of chronic GVHD was 51% [95% CI (38%, 68%)]. The cumulative incidence of relapse within 1 year was 0% [95% CI (0%, 19%)] and 21% [95% CI (11%, 40%)] for patients with early and advanced disease respectively. With a median follow‐up of 28 months (range 16–45 months), 2‐year disease‐free and overall survival for patients transplanted in CR1/CP1 was 81% and 81% [95% CI (64%, 98%)], respectively, and for patients with advanced disease was 33% [95% CI (17%, 50%)] and 40% [95% CI (23%, 57%)] respectively. Complete and persistent donor chimaerism was seen in 77·5% of 40 patients evaluated. All 14 chronic myeloid leukaemia (CML)‐CP1 patients became bcr–abl negative within 250 d. High‐dose ATG pretransplant results in a low incidence of severe aGVHD without compromising donor chimaerism or elimination of minimal residual disease. Our results are similar to data obtained after matched sibling donor transplantation.

Pro-Inflammatory and T Cell Inhibitory Cytokines Are Secreted at High Levels in Tumor Cell Cultures of Human Renal Cell Carcinoma

Objectives: The objectives of this study were to assess cytokine secretion in human renal cell carcinoma (RCC) and to identify cytokines contributing to the immunomodulatory effect of tumor cells. Methods: Cytokine secretion in the supernatant of primary tumor cell cultures (PTCC) and corresponding cell lines (CL) was assayed using ELISA. Tumor cells were characterized by morphology, immunocytochemistry, and flow-cytometric analysis. Tumor-cell-induced T cell activation was determined by coculture of γδ and αβ T cell clones with tumor CL. Results: We assessed the cytokine secretion of tumor cells from 27 PTCC and their corresponding CL (3/27) of RCC. We found that RCC predominantly produced both pro-inflammatory and T-cell-inhibitory cytokines, such as IL-8, IL-6, GM-CSF, TNF-α, IL-10 and TGF-β1. CL were adapted to serum-free medium which may prove as a useful tool in future studies of cytokine secretion in RCC. In addition, we used γδ and αβ T cell clones to assess the immunomodulatory effect of tumor cells from RCC and found that predominantly γδ T cells were activated by RCC. Conclusions: Our data suggest that RCC produce large amounts of both pro-inflammatory and T-cell-inhibitory cytokines that potentially could influence the immune response of the host, especially tumor-specific cytotoxic T cells.

Hematologic and molecular spontaneous remission following sepsis in acute monoblastic leukemia with translocation (9;11): a case report and review of the literature

Abstract:  Spontaneous remission in patients with acute myeloid leukemia (AML) is a rarely reported phenomenon of usually short duration. The etiology remains unclear, but an association with preceding blood transfusions or bacterial infections has been reported. Triggered immune responses are suggested to play a potential role in the development of spontaneous remission. Acute monocytic leukemia was diagnosed in a 61‐yr‐old male patient. Cytogenetic analysis revealed a sole translocation (9;11) (q22;q23) and RT‐PCR the MLL/AF9 fusion gene. As a result of the patients reduced performance status and septic condition, cytostatic therapy was withheld. No microorganisms could be detected. Hematologic and molecular remission occurred after initiating antibiotic therapy without any cytostatic treatment; 29 months after the initial diagnosis, he is in complete remission, and excellent physical condition. Our report includes a review of the literature since 1985, reporting cases of patients with AML and spontaneous remission together with informative cytogenetics. Balanced translocations such as in core binding factor (CBF) leukemias appear somewhat overrepresented. We speculate that AML‐specific T cells might be relevant for induction of spontaneous remission and need to be further investigated.

Myelodysplastic syndrome in transformation to acute myeloid leukemia presenting with diabetes insipidus: due to pituitary infiltration association with abnormalities of chromosomes 3 and 7

Abstract: A 31‐yr‐old woman with myelodysplastic syndrome (MDS) in transformation to acute myeloid leukemia (AML) presented with initial symptoms of polyuria and polydipsia. Cytogenetics revealed monosomy 7 and translocation (3;3)(q21;q26). The initial symptoms, in conjunction with a low serum level of anti‐diuretic hormone (ADH) and magnetic resonance imaging (MRI) findings demonstrating loss of the ‘bright spot’ of the neurohypophysis, indicated diabetes insipidus (DI), e.g. caused by leukemic infiltration of the neurohypophysis. After induction chemotherapy the patients bone marrow revealed blast persistence, and following a second course of chemotherapy and normalisation of MRI, an allogeneic peripheral blood stem cell transplantation (PBSCT) from the patients HLA‐identical brother was performed, resulting in ongoing complete remission. Recently, Lavabre‐Bertrand et al. reported an association of AML with DI, elevated platelet counts, and monosomy 7 and chromosome 3 abnormalities in three patients (Eur. J. Haematol. 2001: 66: 66–69). Our report of an MDS with trilineage dysplasia and these karyotypic changes associated with DI indicates that this new entity may also include preleukemic cases.

Incidence of mixed chimaerism and clinical outcome in 101 patients after myeloablative conditioning regimens and allogeneic stem cell transplantation

In the light of reduced intensity conditioning regimens for allogeneic transplantation, monitoring of donor cell engraftment acquires new relevance. We analysed the clinical significance of haematopoietic chimaerism as a parameter of patient outcome and detection of relapse for early intervention by donor lymphocyte infusion (DLI) after allogeneic transplantation. Between July 1994 and March 1999, 101 adult patients with malignant disease were evaluated. Median follow‐up was 15 months (range 0·7–56·5) after transplantation. Patients received busulphan‐containing (n = 82) or total body irradiation (TBI)‐containing (n = 19) regimens. Fifteen out of 98 (15%) patients with predictive chimaerism analyses relapsed, 5 out of 20 (25%) with mixed chimaerism (MC) and 10 out of 78 (13%) with complete donor chimaerism (CC) before apparent relapse. Seven patients received donor lymphocyte infusions (DLI) as relapse therapy with conversion from MC to CC in all three patients with successful DLI. Our data stress the importance of chimaerism monitoring after allogeneic transplantation and demonstrate the more frequent occurrence of disease relapse in patients showing MC, rather than CC, after transplantation. Moreover, the assessment of chimaerism has been shown to be a valuable tool in monitoring the efficiency of donor lymphocyte infusions for relapse.

Production of insulin-like growth factor binding proteins by human ovarian carcinoma cells.

Cells of the human ovarian carcinoma lines EFO-21, EFO-27, MFO-35 and MFO-36 secrete binding proteins for insulin-like growth factors (IGFBPs) into their culture media. By sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting, seven groups of IGFBPs with molecular masses of 25, 30 (doublet), 34, 37, 40, 45, and 50 kDa were observed, depending on the cell line under investigation. By Northern blot analyses using cDNAs or oligonucleotides specific for the six types of IGFBP (IGFBP-1 to IGFBP-6), mRNA for all IGFBPs tested except for IGFBP-1 could be detected in the ovarian carcinoma cell extracts. In detail, analysis of EFO-21 protein products by SDS-PAGE yielded IGFBPs of 25, 34, and 50 kDa; extracts of EFO-21 cells contained mRNAs for IGFBP-2, -3, -4, and -6. EFO-27 cells produced IGFBPs of 40 kDa and 45 kDa as determined by SDS-PAGE, and mRNAs for IGFBP-3, -4, and -6 were detected. In the conditioned medium of MFO-35 cells, IGFBPs of 25, 30 (doublet), 34, 37, 40, and 45 kDa were observed by SDS-PAGE, while mRNAs for the five proteins IGFBP-2 to IGFBP-6 were found. MFO-36 cells produced IGFBPs of 34 kDa and 50 kDa as determined by SDS-PAGE, and the cells expressed mRNAs for IGFBP-2, -3, -4, and -6. In relation to published molecular mass data of the known IGFBPs, the size of the secreted proteins could be correlated to the mRNA patterns expressed by the ovarian carcinoma cells. It is concluded that ovarian carcinoma cells frequently express IGFBP-3, -4, and -6 and, to a lesser extent, IGFBP-2; the expression of IGFBP-5 appears as a rather rare event, while IGFBP-1 was not found to be expressed in ovarian carcinoma cells.

Comparison of Cytogenetics, Cytokine Secretion, and Oncogene Expression in Primary Cultures of Renal Carcinoma Cells

We compared the cytogenetic pattern of 20 different primary tumor cell cultures (PTCC) of renal cell carcinoma (RCC) to their cytokine secretion and oncogene expression. High secretion of IL-6 (gene locus on chromosome 7p21-p14) was correlated with the gain of an additional chromosome 7. Structural changes involving chromosome 5q22, the site of the GM-CSF gene, were matched with the high secretion of GM-CSF in PTCC. No such association was found for beta 2-microglobulin, TGF-beta 1, TNF-alpha, IL-8, and oncogenes, such as c-fos, c-myc, and pan-ras. Our approach may be useful in simultaneously analyzing several factors contributing to tumor progression and may contribute to understanding the multistep development of RCC.