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: 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

Description: Over the last 1.5 years a number of studies have shown that JAK2 V617F mutations - even if not the initiating mutation of CMPD - are important in particular with respect to diagnosis and prognosis. However, JAK2 mutations have also been described in some cases of AML. To further work out the significance of JAK2 mutations we performed a comprehensive study in comparison to cytogenetics in 1103 CMPD and 271 AML cases. In CMPD the JAK2 mutation rate was 153/179 (85.5%) in polycythemia vera (PV), 123/205 (60.0%) in essential thrombocythemia (ET), 36/60 (73.3%) in chronic idiopathic myelofibrosis (CIMF), and 345/632 (54.6%) in unclassified CMPD matching data of previous studies. Of 73 available karyotypes 18 were aberrant (24.7%). The most frequent cytogenetic aberration in PV was trisomy 9 in 6 of 18 (33%) cases with aberrant karyotypes followed by 20q-deletions (3/18, 17%). In ET in only 5 of 64 (7.8%) cases aberrant karyotypes were detected which showed different non-recurrent aberrations. In CIMF 3/10 had 20q-deletions and 4/10 complex aberrant karyotpes. The AML cohort was unselected and contained a random distribution of karyotypes and FAB subtypes, de novo AML (n=178), s-AML with MDS prephase (n=26), s-AML after CMPD (n=27), t-AML after therapy of a preceeding malignancy (n=39). S-AML after CMPD were defined as an own entity as they may better be regarded as blast crisis of CMPD. The frequency of JAK2 mutations in s-AML after CMPD (16/27, 59.3%) was in the same range as in CMPD overall. A karyotype was available in 15 cases: 3 were normal, 10 complex aberrant (66.7%), five of these contained a trisomy 9 or trisomy 9p, two had other aberrations. No JAK2 mutation was detected in s-AML after MDS. In de novo AML the rate of JAK2 mutated cases was 6.2% (11 of 178 cases). This is in the range of other activating mutations in de novo AML like FLT3-TKD and NRAS mutations. Similarly 2 of 37 (5.4%) pts with t-AML were JAK2 mutated. The pattern of chromosome aberrations in de novo AML was characterized by an extremely high frequency of trisomy 8: 7 of 11 de novo cases with JAK2 mutation (63.6%) had +8, four of these as sole aberration and three in combination with + 9. Two were normal and two complex aberrant (18% each). In comparison, overall 7 of 30 cases (23.3%) with trisomy 8 sole or +8,+9 were JAK2 mutated. Thus JAK2 may be a newly defined cooperating mutation to trisomy 8 whereas a gain of trisomy 9 is known as a typical progression marker in JAK2 mutated cases. Both JAK2 mutated t-AML were t(8;21) positive. In total 21 cases with t(8;21) were analyzed, 3 with t-AML and 18 with de novo AML. Thus 9.5 % of t(8;21) were JAK2 mutated. This may be an event restricted to t-AML. Based on this study we suggest that JAK2V617F in CMPD and AML after CMPD contributes to progression of disease and is followed by chromosomal abnormalities. In CMIF and AML after CMPD most cases have complex aberrant karyotypes suggesting that JAK2 mutation is one step in multistep mutagenesis in which chromosomal instability seems to play a major role. The pathophysiological role in de novo AML seems to be different with JAK2 mutations occurring less frequently overall. They were highly correlated 1) with trisomy 8 in de novo AML and 2) with t(8;21) in t-AML whereas none of 18 de novo AML1-ETO cases were JAK2 mutated. From this we conclude that in AML JAK2 takes over a function as a typical type 1 mutation like FLT3, KIT or RAS at least in combination with +8 or with t(8;21).

Description: After recent reports addressed prognostic factors and outcome in older age AML (Burnett et al. Blood106:162a,2005; Wheatley et al. Blood106:199a,2005; Appelbaum et al. Blood107:3481–5,2006; Farag et al. Blood108:63–73,2006) we evaluated 764 patients of 60–85 (median 66) years reduced to those with de-novo AML, known karyotype, and identical consolidation-maintenance chemotherapy, who were part of the 1992 and 1999 multicenter randomized trials by the German AMLCG (Buchner et al. J Clin Oncol21:4496–504,2003;24:2480–9,2006). 521 patients were 60 -〈 70 (median 64) and 243 patients were 70–85 (median 73) years of age. 64% and 50% patients respectively went into complete remission, 24% and 29% remained with persistent AML, 12% and 21% succumbed to early and hypoplastic death (p

Description: In chronic lymphocytic leukemia (CLL) cytogenetic aberrations such as del(17p) and del(11q) predict inferior outcome. In addition, complex aberrant karyotypes as well as chromosomal translocations as defined by metaphase cytogenetics were suggested as poor prognostic markers for overall survival. We screened 194 consecutive CLL patients for del(17p)/TP53-deletion by fluorescence in situ hybridization (FISH) and for TP53-mutations by denaturing high performance liquid chromatography (DHPLC) and subsequent direct sequencing of aberrant fragments. In addition 160 of these CLL patients were analyzed by classical metaphase cytogenetics to determine the incidence of TP53-aberration in different cytogenetic subgroups. Interphase FISH on 194 samples detected TP53-deletions in 9.3% (n=18) of cases. In parallel, exons 3–9 of the TP53 gene were screened by DHPLC and an aberrant pattern was detected in 9.8% (n=19) of cases. TP53-mutations were confirmed and further characterized by direct sequencing in 16 of the 19 cases. The residual 3 samples had an aberrant pattern in DHPLC for the amplicon of exons 8–9 which pointed to a small population of TP53-aberrant cells which was beyond the detection limit of sequencing. 16 of 18 (89%) cases with TP53-deletion were accompanied by a TP53-mutation affecting the residual allele. 3 samples with TP53-mutations had no deletion of one TP53 allele. Therefore, the overall incidence of TP53-aberrations was 10.8 % (21/194) with a significant association of TP53-deletion and TP53-mutation (p

Description: 13q14 deletions are the most frequent abnormality in CLL and are overall associated with a favourable prognosis. However, the clinical course of the disease is heterogeneous within this subgroup of CLL. In order to characterize this subgroup, which is identified in routine diagnostics by interphase FISH, in more detail we performed chromosome banding analysis in addition. By improving the cultivation technique using the immunostimulatory CpG-oligonucleotide, DSP30, and IL-2 we reached a high success rate in routine diagnostics. Since August 2005 416 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. The FISH panel included probes for the detection of trisomy 12, IGH-rearrangements, and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). 411/416 (98.8%) cases could be successfully stimulated for metaphase generation. 348/411 (84.7%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 332 of 416 (79.8%) successfully evaluated cases. In 229 cases (55%) a 13q14 deletion was detected by FISH, including 58 patients with a homozygous deletion. CBA was not evaluable in 4/229 cases. A normal karyotype was observed in 9/229, due to a small size of the aberrant clone missed by CBA (20% of interphase nuclei) in 1 case and due to the small size of the deletion not visible in CBA in 8 cases (growth of the aberrant clone was confirmed by FISH on metaphases). In 108 cases a deletion 13q was the only abnormality detected in CBA. 29 cases showed one other abnormality in addition to del(13q) (del(11q) n=13, +12 n=2, der(17p) n=3, other abnormalities not detectable by the used FISH panel n=11). In 51 cases 2 or more abnormalities were observed in addition to the 13q deletion. Interestingly, 28 cases did not show a 13q-deletion but a reciprocal translocation or insertion with a breakpoint in 13q14. In all these cases FISH on metaphases was performed with a whole chromosome painting probe for chromosome 13 and a probe for either D13S25 or D13S319, demonstrating a loss of one D13S25/D13S319 signal from the derivative chromosome 13 and the partner. In 9 cases D13S25/D13S25 was also lost from the homologous chromosome 13 (homozygous 13q14 deletion). The translocation partner was confirmed in a second FISH analysis also confirming the reciprocal nature of the abnormality. The breakpoints of the partner chromosomes were distributed all over the genome (1p13, 1q23, 1q24, 1q42, 1q42, 3q21, 3q21, 4p16, 4q23, 5q13, 5q15, 6q11, 6q23, 7p21, 8p23, 8q21, 8q22, 9p22, 9q21, 9q33, 10p15, 10q24, 11p15, 11q23, 13q34, 15q15, 16q24, 16q24). In conclusion, CBA offers important information in addition to interphase FISH in CLL. 1) CBA detects chromosome abnormalities in addition to 13q14 deletion which can not be detected with a standard interphase FISH panel. 2) CBA provides new biological insights into different mechanisms leading to loss of 13q14. Prospective clinical trials have to evaluate the prognostic impact of the different subclasses of CLL with 13q14 deletion that now can be identified by chromosome banding analysis.

Description: Background: Dose intensity is considered one of the prime determinants of antileukemic efficacy in induction treatment of acute myeloid leukemia (AML) - as demonstrated by the superior long-term results of double induction versus conventional induction. In an attempt to further pursue this historically successful strategy an ongoing phase II study of the AML-CG pilots the feasibility of the S-HAM regimen (HD-AraC 3g/m2/12h d1,2,8,9; Mitoxantrone 10mg/m2 d3,4,10,11) in first-line treatment of de-novo AML. In this regimen the interval between the two induction cycles is reduced from 17 days (double induction) to a minimum of 3 days (S-HAM) - thereby increasing dose-intensity more than 2-fold in the critical early phase of treatment. Results: In the past 18 months 99 patients have been recruited into the trial with a median age of 52 years (range 18 – 78). Of 93 patients evaluable for response the following results were achieved: CR 62%, CRp 24%, PL 6%, ED 8% - resulting in an overall response rate (ORR) of 86%. The early death rate (ED) of 8% and the toxicity profile compared favourably with a historical control group within the AML-CG 1999 study (subgroup: de-novo AML, age less than 60 years, HAM-HAM double induction) with 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 88% of patients had a bone marrow blast count of 〈 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. If patients had an adequate blast clearance on day 18 pegylated G-CSF was applied every 10 days until neutrophil recovery. The median time to neutrophil recovery was 30 days after start of treatment with S-HAM which was substantially shorter than following either TAD-HAM or HAM-HAM double induction in the AML-CG 1999 trial (both with a median of 45 days). Since the S-HAM regimen has proven feasible at the present dose level a dose escalation was performed with an additional day of HD-AraC and Mitoxantrone in the first cycle of the sequential regimen. Conclusion: In the future the appropriate dose level of the S-HAM regimen will then constitute the experimental arm for a randomized comparison of a dose-intensified regimen S-HAM - combining a promising antileukemic activity with significantly reduced duration of critical neutropenia - versus standard double induction for patients younger than 60–70 years in the next generation of the AML-CG studies.

Description: Deletions of the long arm of chromosome 5 are typical aberrations in AML and MDS. They occur either as the sole abnormality or within a complex aberrant karyotype. As the precise determination of the breakpoints and the size of the deletion is not possible with chromosome banding analysis alone we performed genomic arrays (Affymetrix 10K arrays) in 32 AML with 5q-deletion within a complex aberrant karyotype and in 17 cases with a 5q-deletion as the sole abnormality (AML: n=3, MDS as typical 5q- syndrome: n=6, other MDS subtypes: n=8). Gene expression analysis using Affymetrix U133A+B or 2.0 plus was performed in addition in all 32 AML cases with complex aberrant karyotype and in 3 cases with 5q deletion sole. The deleted region could be determined based on the genomic array data in 30 of 32 cases with AML and complex aberrant karyotype and in 9 of 17 cases with 5q deletion sole. All evaluable cases showed the 5q deletion in more than 40% of cells (interphase FISH with a 5q31-probe, range 40 to 98%), while less than 45% of interphase nuclei with 5q31-deletion in not evaluable cases (range 8% to 45%). Genomic array analysis mapped the variable proximal breakpoint in the 5q deletion sole group between 5q14.1 and 5q31.3 and the distal breakpoint between 5q32 and 5q35.1. The size of the deletion varied between 27.14 and 81.20 MB (median 73.13 MB). In cases with complex aberrant karyotype the proximal breakpoint was located between 5q11 and 5q23.1 while the distal was located between 5q32 and 5q35.3. The size of the deletion varied between 34.72 and 132.30 MB (median 94.77 MB). Two approaches were tried to determine the size of the deletion based on gene expression data. As a control groups 40 AML and 40 MDS both with normal karyotype were used. Each probe set expressed in at least 1 case of the control group with a precise localiasation on chromosome 5 available was included in the analysis. For each probe set a median expression within the control groups was calculated. First for each probe set a ratio between the individual patient with 5q deletion and the median expression of the respective control group was calculated. For each chromosomal band on chromosome 5 the proportion of genes showing a ratio

Description: In CLL data from chromosome banding analysis have been scarce due to the low proliferative activity in vitro. We improved the cultivation technique using an immunostimulatory CpG-oligonucleotide DSP30 and IL-2 leading to a high success rate of chromosome banding analysis in routine diagnostics. Since August 2005 446 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. Diagnosis of CLL was established by standard criteria based on cytomorphology and immunophenotyping. The FISH panel included probes for the detection of trisomy 12, IGH-rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). 440/446 (98.7%) cases could be successfully stimulated for metaphase generation and are the basis of this study. 370/440 (84.0%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 353 of 440 (80.2%) successfully evaluated cases. Overall 452 abnormalities were detected by FISH and 788 abnormalities by CBA. Based on FISH results 277 cases showed 1, 67 cases 2, 8 cases 3 and 1 case 4 abnormalities, respectively. In CBA at least 1 aberration was detected in 177, 2 in 98, 3 in 45, 4 in 19, and 5 or more aberrations in 31 patients. In 31 of 87 cases (35.6%) showing no aberrations in FISH abnormalities were detected in CBA. On the other hand 14 of 70 cases (20.0%) with a normal karyotype demonstrated abnormalities using FISH. In 7 of these cases CBA missed the abnormalities due to the small size of the aberrant clone or insufficient proliferation of the aberrant clone in vitro and in another 7 cases due to the small size of the 13q deletion not visible in CBA. Using CBA, in total 97 balanced translocations, 169 unbalanced translocations leading to gain and/or loss of genetic material, 368 deletions, 77 gains of whole chromosomes, 40 losses of whole chromosomes, and 37 other aberrations were observed. Only 17 of 97 balanced translocations involved the IGH gene. In 28 cases balanced translocations involved the breakpoint 13q14. Although these translocations were reciprocal and seemed balanced in CBA FISH demonstrated a 13q14 deletion in the breakpoint region. Therefore, based on CBA cases with 13q deletion could be subdivided into 3 different categories: 1. del(13q) sole, 2. del(13q) with additional abnormalities and 3. del(13q) due to a reciprocal translocation. This genetic heterogeneity might account for differences in clinical outcome. In cases with TP53 deletions the number of chromosome abnormalities was higher compared to cases without TP53 deletion (mean 5.0 vs 1.5, p