Claudia Schoch’s research while affiliated with MLL Münchner Leukämielabor GmbH and other places

So far the classification of tumors relies on the interpretation of clinical, histopathological, immunophenotypic, cytogenetic and molecular genetic findings. Especially in hematological tumors a precise analysis of the malignant cells using classical methods such as cytomorphology and histology which both are supplemented by cytochemistry and multiparameter immunophenotyping are used in routine diagnostics for classification. Furthermore, insights into the genetic basis of the disease, i. e. disease-specific chromosomal aberrations and molecular alterations detected in the malignant cell clone, have substantially increased the importance of cytogenetics, fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR) and their combination in establishing the diagnosis in each subentity. In the clinical setting this not only implies a better understanding of the course of distinct disease subtypes but also allows the selection of disease-specific therapeutic approaches, e. g. the use of all-trans retinoic acid (ATRA) in acute promyelocytic leukemia or of imatinib in chronic myeloid leukemia. This is also true for an early application of allogeneic transplantation strategies in AML with complex aberrant karyotypes. Given this genetic background the microarray technology may become an essential tool for the optimization of the classification of tumors and thus may be used as a routine method for diagnostic purposes in the near future.

To evaluate the applicability of NPM1 mutations as follow up markers and to evaluate whether there is any prognostic relevance for NPM1 detection in MRD we studied 531 samples of 130 NPM1 mutated AML during and after therapy. NPM1 mutations are heterogeneous with 80% belonging to type A, 10% to types B and D, and further 10% to more than 40 different yet described rare types. We have established NPM1 specific real time PCR assays for 12 different NPM1 variants. This study included 396 samples (spl) of 98 patients (pts) with type A, 35 spl of 8 pts with type B, 63 spl of 11 pts with type D and 37 spl of 13 pts with 9 different rare types. All assays are RNA based rendering high sensitivities between 1:100,000 to 1:1,000,000. Seven of these assays are specific, while in five of them a minimal detection of the wildtype allele cannot be avoided. Per each patient 2–15 samples were analyzed (mean 4.1, median: 3). Mean follow-up time was 302 days (median: 220 days). The initial expression ratio (% NPM1 mutation/ABL) varied between 21,8 and 8842 (median 540,6). No correlation of this initial ratio to clinical outcome was found. The expression ratios of the NPM1 mutations during follow up correlated well to clinical follow up. In 84 cases with continuing clinical remission (CCR) NPM1 mutation levels were either below traceability or levels were very low or still decreasing. Seven cases were resistant to chemotherapy what was also identified by non-decreasing NPM1 levels. Relapses were detected in 35 cases. At relapse all cases had high NPM1 levels comparable to those at diagnosis. Of these 35 relapses 16 were predictable by increasing NPM1 levels 31–126 days (median 73 days) before clinical relapse. Further eight cases did not reach more than a three log reduction after consolidation and relapsed within the first year after start of therapy. In 11 cases early detection of relapse by PCR was not possible due to lack of samples within the last 6 months before clinical relapse was diagnosed. Four cases had increasing levels at the last follow up with still unclear outcome. Based on these findings 5 different patterns were defined: good response, CCR (n=84), good response, predictable relapse (n=16), < 3 log reduction, followed by relapse (n=8), resistant AML (n=7), non predictable relapse or unclear outcome due to missing MRD samples (n=15). To analyse the impact of NPM1 mutation levels on prognosis four different follow-up intervals were defined: interval 1: day 21–60 after start of therapy; interval 2: days 61–120; interval 3: day 121–365, 4: later. Using Cox regression analysis a significant impact of MRD levels on EFS was detected for interval 1 (p=0.033), interval 2 (p<0.001), interval 3 (p=0.002) and interval 4 (0.037). Furtheron for interval 4 a threshold of 0.01% NPM1/ABL was defined. Thirty cases had a % NPM1/ABL of <0.01% and 28 cases >0.01%. Both groups had significantly different EFS rates (after 2 years: 96.7% vs. 61.5%; p<0.001). In conclusion NPM1 mutations are highly effective and sensitive markers for PCR-based MRD detection in normal karyotype AML, mutation levels during follow up highly correlate with the clinical course of the disease and are highly predictive for prognosis, early detection of relapse is possible by increasing MRD levels up to four month before clinical relapse.

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse-polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5' or 3' of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)-PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Histone deacetylase inhibitor valproic acid (VPA) was recently shown to enhance proliferation and self-renewal of normal hematopoietic stem cells, raising the possibility that VPA may also support growth of leukemic progenitor cells (LPC). Here, VPA maintains a significantly higher proportion of CD34+ LPC and colony forming units compared to control cultures in six AML samples, but selectively reduces leukemic cell numbers in another AML sample with expression of AML1/ETO. Our data suggest a differential effect of VPA on the small population of AML progenitor cells and the bulk of aberrantly differentiated blasts in the majority of AML samples tested.

Application of five-color staining may improve quantification of minimal residual disease by multiparameter flow cytometry in acute myeloid leukemia. We analysed bone marrow samples in 139 cases using a comprehensive antibody panel with five-color combinations. Sensitivity was estimated by quantification of leukemia-associated aberrant immunophenotype (LAIP)-positive cells for each LAIP in 18 normal bone marrow (BM) samples. The logarithmic difference (LD) in LAIP-positive cells between leukemic and normal BM amounted to a median of 3.32 (range 1.76 - 4.89). Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells while percentages of LAIP-positive leukemic cells changed only marginally (median gain in LD = 0.54; maximum gain = 3.30). Because regenerating bone marrow has not been used as control data are most important to post-therapy checkpoints. In 32 patients with clinical follow-up, a LD higher than the median (3.25) at the follow-up checkpoint corresponded to a longer event-free survival. These data suggest that the application of five-color staining significantly improves the sensitivity and accuracy of the method.

Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.

Trisomy 8 is the most frequently observed trisomy in acute myeloid leukemia (AML) occurring as a sole karyotype abnormality or in addition to other chromosome aberrations. It was the aim of this study to analyze the impact of trisomy 8 on the expression of genes located on chromosome 8 in distinct AML subgroups characterized by different chromosome abnormalities in addition to trisomy 8. Gene expression analyses were performed on a total of 567 AML cases comprising the following subgroups: +8 sole, +8 within a complex aberrant karyotype, +8 in addition to t(15;17), inv(16), t(8;21), 11q23/MLL, or other abnormalities, AML with normal karyotype and the before mentioned subgroups without trisomy 8. A significant higher mean expression of genes located on chromosome 8 was observed in subgroups with +8 in comparison to their respective control groups. A varying number of significantly higher expressed genes was identified in all comparisons. No gene was significantly overexpressed in all comparisons, and no distinct gene expression pattern was identified allowing the identification of cases with trisomy 8. In conclusion, the gain of chromosome 8 leads to a higher expression of genes located on chromosome 8. However, no consistent pattern of genes was identified, which shows a higher expression in all AML subtypes with trisomy 8. These data suggest that trisomy 8 rather provides a platform for a higher expression of chromosome 8 genes which are individually up-regulated by the respective primary genetic abnormalities. Therefore, trisomy 8 in AML determines no specific disease characteristic but is a disease modulating secondary event.