Frank Hartmann

Gene mutations and response to treatment with all-trans retinoic acid in elderly patients with acute myeloid leukemia. Results from the AMLSG Trial AML HD98B

The findings of this study suggest that mutant NPM1, and more specifically the genotype mutant NPM1 without FLT3-ITD, is a predictive factor for response to all-trans retinoic acid given as an adjunct to intensive chemotherapy in older patients with acute myeloid leukemia. See related perspective article on page 10. Background In a previous randomized trial, AML HD98B, we showed that administration of all-trans retinoic acid in addition to intensive chemotherapy improved the outcome of older patients with acute myeloid leukemia. The objectives of this study were to evaluate the prognostic impact of gene mutations and to identify predictive genetic factors for the all-trans retinoic acid treatment effect. Design and Methods Data from mutation analyses of the NPM1, CEBPA, FLT3, and MLL genes were correlated with outcome in patients 61 years and older treated within the AML HD98B trial. Results The frequencies of mutations were: NPM1, 23%; CEBPA, 8.5% (analysis restricted to patients with a normal karyotype); FLT3 internal tandem duplications (ITD), 17%; FLT3 tyrosine kinase domain mutations, 5%; and MLL partial tandem duplications, 4.5%. The genotype mutant NPM1 was positively and adverse cytogenetics as well as higher white blood cell count negatively correlated with achievement of complete remission. In Cox regression analysis, a significant interaction between the genotype mutant NPM1 without FLT3-ITD and treatment with all-trans retinoic acid was identified, in that the beneficial effect of all-trans retinoic acid on relapse-free and overall survival was restricted to this subgroup of patients. Other significant factors for survival were age, adverse cytogenetics, and logarithm of white cell count. Conclusions In elderly patients with acute myeloid leukemia, NPM1 mutations are associated with achievement of complete remission, and the genotype ‘mutant NPM1 without FLT3-ITD’ appears to be a predictive marker for response to all-trans retinoic acid given as an adjunct to intensive chemotherapy (ClinicalTrials.gov Identifier: NCT00151242).

Phase III study of all-trans retinoic acid in previously untreated patients 61 years or older with acute myeloid leukemia.

The purpose of our study was (i) to evaluate the impact of all-trans retinoic acid (ATRA) given as adjunct to chemotherapy and (ii) to compare second consolidation vs maintenance therapy in elderly patients with acute myeloid leukemia (AML). A total of 242 patients aged ⩾61 years (median, 66.6 years) with AML were randomly assigned to ATRA beginning on day +3 after the initiation of chemotherapy (ATRA-arm, n=122) or no ATRA (standard-arm, n=120) in combination with induction and first consolidation therapy. A total of 61 patients in complete remission (CR) were randomly assigned to second intense consolidation (n=31) or 1-year oral maintenance therapy (n=30). After induction therapy the intention-to-treat analysis revealed a significant difference in CR rates between the ATRA- and the standard-arm (52 vs 39%; P=0.05). Event-free (EFS) and overall survival (OS) were significantly better in the ATRA-compared to the standard-arm (P=0.03 and 0.01, respectively). OS after second randomization was significantly better for patients assigned to intensive consolidation therapy (P<0.001). The multivariate model for survival revealed lactate dehydrogenase, cytogenetic risk group, age, and first and second randomization as prognostic variables. In conclusion, the addition of ATRA to induction and consolidation therapy may improve CR rate, EFS and OS in elderly patients with AML.

Risk-adapted postremission therapy in acute myeloid leukemia: results of the german multicenter AML HD93 treatment trial

The objective of the AML HD93 treatment trial was to evaluate the outcome in young adults with acute myeloid leukemia (AML) after postremission therapy was stratified according to cytogenetically defined risk. The rationales for the study design were based (i) on previous favorable results with high-dose cytarabine in AML with t(8;21), inv/t(16q22) and in AML with normal karyotype, and ii) on encouraging results obtained in several phase II trials using autologous stem cell transplantation (SCT). Between July 1993 and January 1998, 223 eligible patients, 16–60 years of age with newly diagnosed AML other than French–American–British type M3/M3v, were entered into the trial. Risk groups were defined as follows: low risk: t(8;21) or inv/t(16q22); intermediate risk: normal karyotype; high risk: all other chromosomal abnormalities. Following intensive double induction therapy with idarubicin, cytarabine and etoposide, all patients in complete remission (CR) received a first consolidation therapy with high-dose cytarabine and mitoxantrone (HAM). A second consolidation therapy was stratified according to the risk group: low risk: HAM; intermediate risk: related allogeneic SCT or sequential HAM; high risk: related allogeneic or autologous SCT. Double induction therapy resulted in a high CR rate of 74.5%, and 90% of the responding patients were eligible for consolidation therapy. Survival for all 223 trial entrants was 40%, and for the 166 patients who entered CR, disease-free (DFS) and overall survival were 40 and 51% after 5 years, respectively. Within the low-, intermediate- and high-risk groups, DFS and survival after 5 years were 62.5 and 87, 40 and 49 and 17 and 26% respectively, without advantage for allogeneic transplantation in the intermediate- and high-risk groups. Postremission therapy-related mortality was 0, 7 and 14%, respectively. This study demonstrates the feasibility of cytogenetically defined risk-adapted consolidation therapy. The overall trial results are at least equivalent to those of published trials supporting the risk-adapted treatment strategy.

Effects of the tyrosine‐kinase inhibitor geldanamycin on ligand‐induced HER‐2/NEU activation, receptor expression and proliferation of HER‐2‐positive malignant cell lines

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine‐kinase inhibitors. We have examined its effects on Her‐2/neu kinase activity, protein expression level, and proliferation of Her‐2+ malignant cells. In SK‐BR‐3 breast‐cancer cells, short‐time treatment with geldanamycin completely abrogated gp30‐ligand‐induced activation of Her‐2 without a change of receptor‐expression level. Longer treatment of intact cells with geldanamycin induced decreased steady‐state Her‐2 autophosphorylation activity, which correlated with reduction of Her‐2 protein expression and phosphotyrosine content of several proteins. The decrease was time‐ and dose‐dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her‐2 protein level probably resulted from increased degradation, since the Her‐2 mRNA level remained constant. Geldanamycin effects were not specific for Her‐2, since the non‐receptor tyrosine‐kinase fyn was inhibited equally. In contrast to these results, protein‐kinase‐C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her‐2 (SK‐BR‐3 > SK‐OV‐3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her‐2‐kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]‐thymidine‐uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK‐BR‐3 (IC50 2nM) and MCF7 (IC50 20nM), while OVCAR3 was only moderately sensitive (IC50 2μM) and SK‐OV‐3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced. Int. J. Cancer, 70:221–229, 1997.

Intensive consolidation versus oral maintenance therapy in patients 61 years or older with acute myeloid leukemia in first remission: results of second randomization of the AML HD98-B treatment trial

Intensive consolidation versus oral maintenance therapy in patients 61 years or older with acute myeloid leukemia in first remission: results of second randomization of the AML HD98-B treatment Trial

Naturally occurring T-cell response against mutated p21 ras oncoprotein in pancreatic cancer.

Mutated p21 ras proteins (muRas) are present in approximately 90% of pancreatic adenocarcinomas and express mutants which can function as cancer-specific antigens. To evaluate the frequency and magnitude of the natural T-cell response against muRas in 19 HLA-A2-positive patients with muRas-positive pancreatic carcinomas, antigen-experienced T lymphocytes in fresh peripheral blood mononuclear cells were shown by IFN-gamma enzyme-linked immunospot using muRas peptides (5-21) that encompass both HLA class I (HLA-A2)- and class II-restricted (HLA-DRB1) epitopes. Six of 19 patients (32%) were found to have a specific T-cell response against individual mutation-specific ras(5-21) but not against other ras mutations or wild-type ras. In contrast, none of 19 healthy subjects had T cells specifically secreting IFN-gamma (P = 0.004). The T-cell response consisted of both CD8(+) and CD4(+) T cells but was dominated by CD8 T cells in three of four patients. MuRas(5-14) and muRas(6-14) were shown to specifically induce CD8(+) T-cell mediated cytotoxicity against HLA-A2-positive, muRas-bearing pancreatic carcinoma cells. The T-cell response was not correlated with prognostic or clinical variables such as tumor-node-metastasis status, stage, or survival. In conclusion, a natural T-cell response against muRas proteins that could be exploited for immunostimulatory therapeutic approaches has been shown in a significant proportion of patients with pancreatic cancer.

Successful collection of peripheral blood progenitor cells in patients with acute myeloid leukaemia following early consolidation therapy with granulocyte colony-stimulating factor-supported high-dose cytarabine and mitoxantrone.

We evaluated the feasibility of collecting peripheral blood progenitor cells (PBPC) in patients with acute myeloid leukaemia (AML) following two cycles of induction chemotherapy with idarubicin, cytarabine and etoposide (ICE), and one cycle of consolidation therapy with high‐dose cytarabine and mitoxantrone (HAM). Thirty‐six patients of the multicentre treatment trial AML HD93 were enrolled in this study, and a sufficient number of PBPC was harvested in 30 (83%). Individual peak concentrations of CD34+ cells in the blood varied (range 13.1–291.5/μl; median 20.0/μl). To reach the target quantity of 2.5 × 106 CD34+ cells/kg, between one and six (median two) leukaphereses (LP) were performed. The LP products contained between 0.2 × 106 and 18.9 × 106 CD34+cells/kg (median 1.2 × 106/kg). Multivariate analysis showed that the white blood cell count prior to HAM and the time interval from the start of HAM therapy to reach an unsupported platelet count > 20 × 109/l were predictive for the peak value of CD34+ cells in the blood during the G‐CSF stimulated haematological recovery. In 16 patients an intraindividual comparison was made between bone marrow (BM) and PBPC grafts. Compared to BM grafts, PBPC grafts contained 14‐fold more MNC, 5‐fold more CD34+ cells and 36‐fold more CFU‐GM. A CD34+ subset analysis showed that blood‐derived CD34+ cells had a more immature phenotype as indicated by a lower mean fluorescence intensity for HLA‐DR and CD38. In addition, the proportion of CD34+/Thy‐1+ cells tended to be greater in the PBPC grafts. The data indicate that sufficient PBPC can be collected in the majority of patients with AML following intensive double induction and first consolidation therapy with high‐dose cytarabine and mitoxantrone.

Anti-CD16/CD30 bispecific antibodies as possible treatment for refractory Hodgkin's disease.

Fifteen patients with refractory Hodgkins disease were treated in a phase I/II dose escalation trial with the NK-cell activating bispecific monoclonal antibody HRS-3/A9 which is directed against the Fcgamma-receptor III (CD16 antigen) and the Hodgkins associated CD30 antigen, respectively. HRS-3/A9 was given four times every 3-4 days starting with 1 mg/m2. The treatment was well tolerated and the maximum tolerated dose was not reached at 64 mg/m2, the highest dose given due to limited amounts of HRS-3/A9 available. Mild to moderate side effects occured in six patients and consisted of fever, pain in involved lymph nodes, and a maculopapulous rash. Median counts of NK-cells and of all lymphocyte subsets were considerably decreased in the patients before therapy and showed no consistent changes under therapy. Eight patients developed human anti-mouse immunoglobulin antibodies, and five patients showed an allergic reaction after attempted retreatment. One complete and one partial remission (lasting 6 and 3 months, respectively), three minor responses (lasting 1 to 15 months), two disease stabilizations (for 2 and 17 months, respectively), and one mixed response were achieved. There was no clearcut dose-side effect or dose-response correlation. Our results encourage further clinical trials with this novel immunotherapeutic approach and emphasize the necessity to reduce the immunogenicity of the murine bispecific antibodies.

Isolation and analysis of two strongly transforming isoforms of the Epstein‐Barr‐virus(EBV)‐encoded latent membrane protein‐1 (LMP1) from a single Hodgkin's lymphoma

Two genes encoding the latent membrane protein 1 (LMP1) of the Epstein‐Barr virus (EBV) were isolated from a single case of Hodgkins disease (HD) and were tested for their biological activities. The LMP1 gene from the Reed‐Sternberg cells contained point mutations relative to the prototype LMP1 gene, leading to amino‐acid exchanges. The LMP1 gene from passenger lymphocytes showed identical point mutations, but also had an in‐frame insertion of 132 base pairs within the 33‐bp repeat region. This insert encoding 44 amino acids contained the sequence PSQQS, corresponding to the potential TRAF‐binding motif PXQXT/S. When compared to the B95.8 gene, both HD‐derived LMP1 genes showed an increase in the transformation of Rat‐1 rodent fibroblasts. The transforming ability of the LMP1 gene with the insertion was greater than that of the other HD‐derived LMP1, and was comparable with the highly transforming LMP1‐Cao gene derived from a nasopharyngeal carcinoma. The HD‐derived genes stimulated expression of the cell‐surface markers, CD40 and CD54, similarly to the LMP1‐B95.8 gene, while the LMP1‐Cao gene had a significantly reduced ability to induce these proteins. In contrast, the LMP1‐Cao transactivated an NF‐κB‐response element more efficiently than did the HD‐derived genes. Transfer of the 132‐bp insert alone into the B95.8 gene did not increase its transforming activity to the LMP1‐Cao level, indicating that additional mutations in the LMP1 gene are necessary for modulating this function. Int. J. Cancer 76:194–200, 1998.© 1998 Wiley‐Liss, Inc.

Initiation of humoral and cellular immune responses in patients with refractory Hodgkin's disease by treatment with an anti-CD16/CD30 bispecific antibody

Abstract Fifteen patients with refractory Hodgkins disease were treated in a dose-escalation trial with the bispecific monoclonal antibody (bi-mAb) HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkins-associated CD30 antigen. Treatment consisted of four cycles of four bi-mAb infusions given over 1 h every 3–4 days at different dose levels ranging from 1 mg/m2 to 64 mg/m2. Measurable serum levels (above 0.1 μg/ml) of circulating bi-mAb could be detected in patients treated with doses above 4 mg/m2, reaching peak levels of 9.5 μg/ml immediately after the end of antibody infusion on the highest dose level. Bi-mAb elimination corresponded to second-order kinetics with a terminal half-life time (t1/2,β) of 28–32 h. Bi-mAb treatment induced the occurrence of human anti-(mouse Ig) antibodies (HAMA) in 6 out of 13 patients initially testing negative. All 6 patients not only developed anti-isotypic anti-(mouse Ig) but also anti-idiotypic and anti-anti-idiotypic antibodies. While no consistent changes of peripheral blood cell counts, or of any lymphocyte subpopulation including natural killer (NK) cells, has been observed, 4 out of 6 evaluable patients treated with doses of at least 4 mg/m2 showed an increase of NK cell activity within 2 weeks after treatment, which lasted for a maximum of 12 weeks. Circulating amounts of soluble CD30 antigen could be detected in the serum of 6 patients. However, like the results and time courses of all the other immunological parameters evaluated, this was not predictive for treatment outcome.