ALBERT

Description: The prognostic value of MRD detection in AML has been shown for PML-RARA, AML1-ETO, and CBFB-MYH11positive AML, a group which accounts for only 20–25% of AML. The largest group of AML is represented by cases with normal karyotype or unbalanced intermediate risk group karyotypes. In this group different molecular mutations occur, which are associated with heterogeneous clinical outcome. Thus, different MRD patterns can be anticipated. To expand the spectrum of molecular targets in intermediate risk AML, we used MLL-PTD, FLT3-LM, and NPM1 which are detectable in 45–50% of all intermediate risk group pts for MRD detection. In addition, overexpression of WT1 for MRD detection was used to even include pts. without detectable mutations. In total 996 samples (spl) of 234 patients (pts) were analysed by quantitative real time PCR. For MLL-PTD (321 spl of 78 pt), and WT1 (336 spl of 66 pts) universal assays were used. For NPM1 mutation specific assays (182 spl of 54 pts (42 x type A, 2 x type B, 7 x type D, 3 x rare not yet defined types)) and for FLT3-LM (161 spl of 18 selected pts) patient specific assays were used. All assays were RNA based. Sensitivity of the assays were between 1:100.000 to 1:1.000.000 for the FLT3-LM and NPM1, depending on pts specific assay. Sensitivity was 1:10.000 to 1:100.000 for MLL-PTD due to low background levels detectable in healthy controls. The sensitivity of WT1 was relatively low with 1:100 at most, as there was no high WT1 expressor at diagnosis in this cohort. With all markers the clinical course of the disease was clearly be reflected and all 84 relapses were detectable due to recurring high expression rates. In 17 cases, were samples 2–4 months before clinical relapse were available relapses were predictable based on increasing transcript levels. Five different follow up intervals (int) were defined: up to day 21; days 22–60; days 61–120; day 121–365, later. The log change from diagnosis to defined follow up intervals was analyzed. A rapid decline of median transcript ratios in the NPM1 group (int 1: 2 log; int 2 to 4: 4 log) was observed. Relapses in the NPM1 group occurred earliest after one year. AML with FLT3-LM similarly showed good responses with 1–2 log decreases in int 1 and 2 and 3 log in int 3 and 4. Also this group revealed the first relapses in int 5. The MLL-PTD group was characterized by slow response rates with only 0.2 log reduction in int 1 and 2 and hardly 3 log in int 3. In this group many relapses occurred in int 4 and 5. These data reflect the biological differences of these molecular subgroups: NPM1 as a favourable group, FLT3-LM as a slightly unfavourable group with good response rates but high relapse rates, and MLL-PTD as an entity with bad prognosis due to poor response rates and high relapse rates. Due to low sensitivity WT1 reflected only 0.2 log in interval 1 up to 2 log in interval 3. Seven cases were analyzed in parallel for WT1 and MLL-PTD, 6 for WT1 and FLT3-LM, and one for FLT3-LM and MLL-PTD. Although the correlation of parallel assessment was high (R=0.993) the median differences of log changes of FLT3-LM and NPM1 was one log larger than for MLL-PTD and three log larger than for WT1, depending on the initial sensitivity of the assays. In conclusion: MRD detection is feasible in the karyotypically intermediate risk group. it nicely reflects biological difference in this group NPM1, MLL-PTD and FLT3-LM are better MRD-markers than WT1, which may only be investigated in cases without any other available marker.

Description: Dose density during early induction has been demonstrated to be one of the prime determinants for antileukemic efficacy. The German AML-CG therefore pilots a dose dense induction regimen S-HAM (sequential HD-AraC [3g/m2/12h d1,2,8,9] and Mitoxantrone 10mg/m2 [d3,4,10,11] followed by pegfilgrastim) in which two induction cycles are applied over 11 – 12 days as compared to conventional double induction, in which two cycles are applied over 25 – 29 days - thereby increasing dose density ca. two-fold in the critical first weeks of treatment. In the past 2,5 years 168 patients with de-novo AML (excluding APL) have been recruited into the trial with a median age of 53 years (range 18 – 78). Of 136 patients evaluable for response the following results were achieved: CR 62%, CRi 22%, PL 7%, ED 9% - resulting in an overall response rate (ORR) of 84%. The early death rate (ED) of 9% and the toxicity profile compared favourably with a historical control group of the AML-CG 1999 study (de-novo AML, 〈 60 years, HAM-HAM double induction) which demonstrated an ED rate of 14% (ORR 68%, persistent leukemia (PL) 18%). The high antileukemic efficacy of S-HAM was also demonstrated by the fact that 89% of patients had a blast count of 〈 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. Whereas even for patients with unfavourable cytogenetics (including complex aberrations) a median overall survival of 13,5 months was reached (23% at 2 years), for patients with favourable karyotypes overall survival at 2 years was 81%and for patients with intermediate karyotypes 74% after S-HAM treatment. Importantly the compression of the two induction cycles into the first 11 – 12 days of treatment seems actually beneficial for normal hematopoesis as demonstrated by a significantly shortened duration of critical neutropenia of 30 days as compared to 45 days after conventionally timed double induction. This shortening of critical neutropenia by more than 2 weeks was highly relevant for the duration of hospital stay and hospital costs. In conclusion S-HAM with pegfilgrastim support is a highly effective regimen in primary de-novo AML with a very favourable safety profile and significantly shortened duration of neutropenia. This regimen will therefore constitute the (dose-dense) experimental arm for a randomized comparison with standard double induction in the next generation of the German AML-CG studies.

Description: Recent data indicate that mutations in exon 12 of the nucleophosmin (NPM1) gene characterize a distinct subgroup of adult and pediatric acute myeloid leukemia (AML). AML carrying NPM1 mutations account for about one-third of all adult AML, exhibit distinctive biological and clinical features and show a strong association to AML with normal karyotype (55% mutated). However, the role of NPM1 in leukemogenesis still remains elusive. Here we present data on a cohort of n=66 AML cases with normal karyotype analyzed by high-density whole genome expression microarrays (Affymetrix HG-U133 Plus 2.0). In parallel melting curve analysis was used to assess NPM1 mutational status: 41 cases were characterized as mutated (NPM1+) and 25 cases were unmutated (NPM1−). We first investigated the gene signature that discriminated NPM1+ from NPM1− cases. Genes that were significantly overexpressed comparing NPM1+ against NPM1– cases included a strong homeobox genes signature (HOXA1, HOXA5, HOXA7, HOXA9, HOXA10, HOXA11, HOXB2, HOXB4, HOXB5, HOXB6, HOXB7, MEIS1, and PBX3). A functional analysis (Gene Ontology) revealed a clear association of the group of overexpressed genes with the cell components nucleosome, chromatin, and the nuclear envelope-endoplasmatic reticulum network as well as involvement in the biological processes of nucleosome and chromatin assembly, establishment of protein transport and localization, and Notch signaling pathway. In contrast, the cellular processes completely differed when genes were investigated that were significantly underexpressed in NPM1+ cases compared to NPM1− cases. This group of genes encoded membrane-related proteins (gap junction, intercellular junction, signalosome complex) and proteins involved in cellular morphogenesis and cell communication. The differences in gene expression signatures between NPM1+ and NPM1− cases permit a robust classification approach by gene expression profiling. Support Vector Machine analysis resulted in 〉92% prediction accuracy of NPM1 mutation status (10-fold cross-validation). The sensitivity was very high for the positive detection of NPM1+ cases (〉97%). Using a 100-fold re-sampling approach and splitting the complete data set into a training set (n=44) and testing set (n=22) the following genes were most frequently selected as top discriminatory genes: HOXA5, HOXB4, HOXB5, HOXB6, MEIS1, PBX3, FGFR1, ADAM17, PRICKLE1, and TMPO. Interestingly, the classification was less accurate when also FLT3 internal tandem duplication mutation status was taken into account. The study cohort (n=66) then was distributed as follows: 19 NPM1+/FLT3+, 22 NPM1+/FLT3−, 4 NPM1−/FLT3+, and 21 NPM1−/FLT3− negative cases. Only 14 of 22 (64%) NPM1+/FLT3– cases were correctly predicted, with miscalls falling both into the group of NPM1+/FLT3+ and NPM1−/FLT3− cases. In conclusion, NPM1 mutations are the most frequent mutations in adult AML to date and their central prognostic role is increasingly recognized. Given the fact that they are nearly mutually exclusive with major recurrent genetic abnormalities and that they can be characterized by a distinctive gene expression program these data especially for of NPM1+/FLT3− AML with better outcome may support to classify this as a separate biological subgroup of AML with normal karyotype.

Description: Up to 95% of all patients (pts) with polycythemia vera (PV) carry JAK2V617F mutations (V617Fmut). The underlying pathophysiologic event in the remaining 5% remained unclear. Recently, novel mutations in exon12 (exon12mut) of the JAK2 gene have been described. Thus far, 16 pts with 4 different mutation types (F537-K39delinsL, H538QK539L, N542-E543del, and K539L) have been identidied by two groups. A specific phenotype of isolated erythrocytosis and young age has been suggested by one group while another group found that besides pts with isolated erythrocythosis around 50% may present with typical criteria for PV. Thus, exon12mut are scarce with still unclear frequency. Therefore, we have applied melting curve screening covering codons 535-555 of exon12 on 211 pts with PV or suspected PV. DNA of all pts with aberrant curve was sequenced. We detected exon12mut in 10/211 (4.7%) of all pts. Of these 99 pts fulfilled diagnostic criteria of PV whereas 112 were suspected PV due to unclear polyglobulia. The frequency of exon12mut in V617Funmut (V617wt) PV was 10/99 pts (10.1%). No exon12mut was detected in pts with polyglobulia. In addition, 10 V617wt ET and 50 CMPD with elevated red blood cell counts were analyzed but turned out to be exon12 unmutated (exon12wt). 3 of the exon12mut pts had the previously described F537-K39delinsL, 2 the N542-E543del, 1 H538QK539K, 1 K539L. In addition, 2 new mutations were detected: a E543-D544del (2 pts) and a K539S mutation (1 pt). Quantification showed exon12mut in 10–30% of all PBC (7 pts) or BM cells (3 pts). 5 pts were analyzed at diagnoses and 5 pts 2–8 years (y) (median 4y) after initial diagnosis of PV. Treatment was phlebotomy and ASS only in all pts. In 1 pt a homozygous K539L mutation was found. Thus, unlike previously reported, the exon12mut like the V617F mutations can occur in a homozygous state. This pt was at advanced stage near to transformation to AML. While in PV overall the female/male ratio was 225/231 in the exon12 mutated cohort the female/male ratio was higher with 7/3. Age of onset was 16–75 y (median: 57.5 y). This is the same range as the exon12wt/V617wt pts (57.3 y) but younger than the V617Fmut PV (66.5 y). Hematocrit of the exon12mut pts was elevated with a median of 61% in males and 55% in females. Erythropoietin levels were very low with a median of 3.8 U/L. Other parameters were less conspicuous in exon12mut than in V617Fmut (given in medians): WBC: 6,100 vs. 12,200/μl; Hb: 150 vs. 158 g/l; platelets 282,000 vs. 483,000/μl. In detail, four pts had elevated WBC and platelets in addition to erythrocytosis whereas 6 pts had isolated erythrocytosis. Thus, exon12mut pts frequently show isolated polyglobulia (60%) which is rare in V617Fmut PV. In addition, none of the exon12mut pts had hepatomegaly, three pts had borderline splenomegaly. In conclusion, analysis for JAK2 exon 12 mutations is a new tool for diagnostics of PV. Based on this still small cohort it can be speculated that exon12mut in comparison to V617Fmut pts tend to occur more frequently in women, at younger age, have lower WBC and platelet counts and frequently isolated erythrocytosis.

Description: In chromic myeloproliferative diseases (CMPD) CML can be identified by the presence of a BCR-ABL fusion gene. The genetic spectrum of the BCR-ABL negative disorders is diverse. The most frequent aberrations especially in PV, ET, and CIMF is the JAK2V617F mutation. Initially these two aberrations were considered to occur mutually exclusive and define different diseases. However, recently 3 reports documented coincidences of the JAK2V617F and BCR-ABL in individual cases. For further clarification we analyzed a cohort of 2317 cases which were screened for BCR-ABL and JAK2 in parallel due to suspected CMPD. Within this cohort 1249 (53.9%) were BCR-ABL-/JAK2V617F-, 119 (5.1%) were BCR-ABL+/JAK2V617F-, and 945 were BCR-ABL-/JAK2V617F+ (40.8%). Double positivity for BCR-ABL and JAK2V617F was detected in 4 cases (0.17%). Real-time PCR for BCR-ABL expression and the V617F was performed for all available timepoints during follow-up. In 2 cases only one timepoint was available and 2 pts were followed for 1 year and 7 months, respectively. After retrospective analysis of the clinical data different patterns of the mutations were detected: 1 pt: A 56-year old male patient was diagnosed with CML in chronic phase in 9/2004. Imatinib treatment led to a major molecular response, but in 10/2006 a marked thrombocytosis led to the diagnosis of a JAK2V617F positive CMPD. Retrospective real-time quantification revealed that the JAK2V617F stayed at the same level as was detected at diagnosis of CML and during one year of follow up whereas the %BCR-ABL/ABL decreased during treatment with imatinib over three log ranges. This indicated that the two mutations occurred in separate clones. 2 pt: A 45 year old male was diagnosed with CML in chronic phase in 9/2006. After 7 months of imatinib treatment RQ-PCR showed decrease of %BCR-ABL/ABL to 0.225 but JAK2V617F remained at a 30% level. This case resembled the first case and suggests different clones - one with BCR-ABL fusiongene and one with JAK2V617F. 3 pt: A 75-year old female, was diagnosed with BCR-ABL negative CMPD in 11/2000. Cytoreduction by hydroxurea (HU) was successful from 2004–2005, but in May 2006 the disease showed signs of acceleration. Molecular diagnostics showed a JAK2V617F and for the first time a BCR-ABL fusion gene, both with expression levels typical for untreated cases. Imatinib was not tolerated, and HU was started again. Over 7 months %BCR-ABL/ABL and %JAK2V617F/ABL remained at the same high level suggesting that one clone harbored both mutations. 4 pt: A 53 years old female was diagnosed with CMPD. 21 years later in 6/2005 molecular diagnostics was performed upon increasing thrombocytosis and showed BCR-ABL positivity and a homozygous JAK2V617F. Both mutations had high expression levels which persisted after 12 months of imatinib treatment. As in the third case the homozygous JAK2 mutational status and the high BCR-ABL expression suggested coexistence of both mutations in the same clone and resistance to imatinib. In conclusion, these four new cases support the idea that BCR-ABL and JAK2 mutations can occur in same patients in the same clone or in different clones and at different time points. It may be speculated that in these cases there is an unknown common antecedent defect.

Description: Dysplastic features can be detected in different cell lineages in myelodysplastic syndromes (MDS) by multiparameter flow cytometry (MFC). The aim of the present study has been the assessment of the flow cytometric detection of dysplastic features previously published to occur in MDS in relation to findings in cytomorphology (CM) and cytogenetics (CG). We analyzed 307 bone marrow samples from patients with suspected (n=130) or proven (n=177) MDS by MFC, CM, and CG in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 3.5%) and from 0% to 23% (median, 3%; r=0.271, p

Description: CLL is a genetically heterogeneous disease. Genetic aberrations allow to distinguish different biological subgroups within CLL. Based on chromosome banding analysis we identified complete and partial gain of the short arm of chromosome 2 always including 2p23 to 2p25 in 28/1051 CLL cases (2.7%) as a new recurring chromosome aberration. Recurring aberrations accompanying gain of 2p were: loss of 1p (n=3), 1q (n=3), 2q (n=3), 6q (n=3), 8p (n=7), 11q (n=10), 12q (n=4), 13q (n=21), 17p (n=7), 17q (n=3), 18p (n=5), and gain of 2q (n=3), 3q (n=3), 13q (n=3), 21q (n=3). In 24/28 cases the mutational status of the immunoglobulin variable heavy chain gene (IgVH) was available. 20 cases showed an unmutated and only 4 a mutated IgVH status. Thus, 2p gain is significantly associated with an unmutated IgVH status as compared to the non 2p group (83% vs 51%, p=0.002). In 8 cases an ATM deletion (29%) and in 5 cases a TP53 deletion (18%) (1 case showed both) were observed (frequency in non 2p+ cohort: 12%; p=0.036 and 7%; p=0.031). A median number of 4 chromosome aberrations per case was observed in 2p+ CLL (range: 1–16, mean=5.2) as compared to only 1 abnormality per case in the non 2p+ cohort (range: 0–10, mean 1.7) (p

Description: The t(11;14)(q13;q32) is considered a marker of mantle cell lymphoma (MCL), while the t(14;18)(q32,q21) is associated with follicular lymphoma. However, both translocations have also been reported in other B-cell malignancies. We evaluated immunophenotypic characteristics as well as cytogenetic aberrations occurring in association with t(11;14) and t(14;18) in order to define specific entities. 51 B-cell lymphoproliferative disorders with t(11;14) and 26 with t(14;18) were studied by chromosome banding, FISH, and immunophenotyping. Based on immunophenotyping 13 of 26 t(14;18)+ cases were classified as NHL and 13 cases as CLL (9 CLL and 4 CLL/PL). The mean number of cytogenetic aberrations observed in addition to t(14;18) was 1.1 in CLL cases and 4.2 in NHL cases (p=0.016). While 9 of 13 B-NHL cases (69.2%) revealed a complex karyotype (≥3 aberrations in addition to t(14;18)), none of the 13 CLL cases did (p=0.0002). In CLL the only recurring aberrations occurring in addition to t(14;18) were +12 (n=7) and −13q (n=5). In B-NHL cases additional aberrations were +1q (n=3), –4q (n=2), –6q (n=5), +7 (n=3), +12 (n=4), –15q (n=2), +der(18)t(14;18) (n=2), +21 (n=2), +22 (n=2), and +X (n=2). Notably, deletion 13q was not detectable in t(14;18)+ B-NHL compared to t(14;18)+ CLL (n=5) (p=0.044). Two antigens were significantly higher expressed in t(14;18)+ B-NHL as compared to t(14;18)+ CLL: CD10 (mean positivity (mp) 34.9% vs. 1.7%, p=0.006), and CD38 (60.9% vs. 29.8%, p=0.014), while 3 antigens were significantly lower: CD11c (38.6% vs. 14.5%, p=0.005), CD23 (66.6% vs. 24.0%, p=0.001), and CD5 (80.8% vs. 29.5%, p

Description: Compared with fluorescence in situ hybridization (FISH), conventional metaphase cytogenetics play only a minor prognostic role in chronic lymphocytic leukemia (CLL) so far, due to technical problems resulting from limited proliferation of CLL cells in vitro. Here, we present a simple method for in vitro stimulation of CLL cells that overcomes this limitation. In our unselected patient population, 125 of 132 cases could be successfully stimulated for metaphase generation by culture with the immunostimulatory CpG-oligonucleotide DSP30 plus interleukin 2. Of 125 cases, 101 showed chromosomal aberrations. The aberration rate is comparable to the rate detected by parallel interphase FISH. In 47 patients, conventional cytogenetics detected additional aberrations not detected by FISH analysis. A complex aberrant karyotype, defined as one having at least 3 aberrations, was detected in 30 of 125 patients, compared with only one such case as defined by FISH. Conventional cytogenetics frequently detected balanced and unbalanced translocations. A significant correlation of the poor-prognosis unmutated IgVH status with unbalanced translocations and of the likewise poor-prognosis CD38 expression to balanced translocations and complex aberrant karyotype was found. We demonstrate that FISH analysis underestimates the complexity of chromosomal aberrations in CLL. Therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.

Description: The TP53 gene is the most frequently mutated gene in human tumors identified so far. In a prior study we demonstrated that 78% of AML with complex aberrant karyotype show a mutation of the TP53 gene. The aim of this study was to determine the frequency of TP53 mutations in an unselected cohort of AML and to analyze the relation to cytogenetic and molecular genetic aberrations. In total 149 AML cases were examined by chromosome banding analysis and screened for FLT3-length mutations (FLT3-LM), MLL partial tandem duplication (MLL-PTD), NPM1 mutations, and TP53 mutations. The cohort included cases with t(8;21) (n=10), t(15;17) (n=6), inv(16) (n=4), 11q23/MLL-rearrangement (n=6), trisomy 8 sole (n=13), AML with normal karyotype (n=46), AML with complex aberrant karyotype defined as showing 3 and more clonal abnormalities but no balanced rearrangement leading to a leukemia specific fusion gene (n=26), and AML with other abnormalities (n=38). FLT3-LM were observed in 21, MLL-PTD in 4, and NPM1-Mutations in 26 cases. TP53 mutation screening of exons 3–9 was performed by denaturing high performance liquid chromatography (DHPLC). All mutations detected were verified by direct sequencing. Overall, TP53 mutations were detected in 20 of the 149 cases (13.4%). Within this cohort of TP53 mutated cases, coincidences of FLT3-LM and MLL-PTD, respectively, with TP53 mutation were detected in one case each. A complex aberrant karyotype was present in 17 of 20 cases (85%) with TP53 mutation. The remaining 3 cases with TP53 mutation showed a normal karyotype, a trisomy 8, and t(8;21) as the sole abnormality, respectively. Therefore, we confirmed a high incidence of TP53 mutations in AML with complex aberrant karyotype (17/26, 65.4%). On the other hand TP53 mutations are very rare in AML without a complex aberrant karyotype (3/123, 2.4%). Furthermore, we divided AML with complex aberrant karyotype into two subgroups:AML with “typical” complex aberrant karyotype showing a deletion of at least one of the following regions: 5q31, 7q31, 17p13 (definition according to Schoch et al. GCC, 2005) andAML with “untypical” complex aberrant karyotype comprizing all others. Interestingly, the frequency of TP53 mutations within the “typical” complex aberrant karyotype group was 75% (15/20) while in the “untypical” group it was 33% (2/6) (p=0.138). In conclusion, the overall incidence of TP53 mutations is low in AML. TP53 mutations are highly associated with AML and complex aberrant karyotype and occur very infrequently in all other cytogenetic subgroups (p