Identification of prognostic parameters in CLL with no abnormalities detected by chromosome banding and FISH analyses.

Chronic Lymphocytic Leukaemia (CLL) is a heterogeneous disease with a clinical course dependent on cytogenetic features. However, in 15-20% of cases both chromosome banding and fluorescence in situ hybridisation analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we performed a comprehensive analysis on 171 patients including genomic arrays (comparative genomic hybridisation and single nucleotide polymorphism), immunoglobulin heavy chain variable region genes (IGHV) status, flow cytometry and targeted sequencing. Genomic arrays detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV status was unmutated in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (15/171; 9%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of these, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, by integrating genomic and molecular data, we identified patients at higher risk for treatment need.

Gain of chromosome 21 or amplification of chromosome arm 21q is one mechanism for increased ERG expression in acute myeloid leukemia.

In acute myeloid leukemia (AML), acquired genomic gains and losses are common and lead to altered expression of genes located within or nearby the affected regions. Increased expression of the ETS-related transcription factor gene ERG has been described in myeloid malignancies with chromosomal rearrangements involving chromosome band 21q22, but also in cytogenetically normal AML, where it is associated with adverse prognosis. In this study, fluorescence in situ hybridization on interphase nuclei disclosed an amplification of the ERG gene (more than six copies) in 33 AML patients with structural rearrangements of 21q22. Array comparative genomic hybridization of these cases disclosed a minimal amplified region at the position 39.6-40.0 Mbp from pter that harbors ERG as the only gene. Analysis by quantitative real-time reverse transcription polymerase chain reaction revealed significantly higher ERG mRNA expression in these patients and in a group of 95 AML patients with complete or partial gain of chromosome 21 (three to six copies) compared with 351 AML patients without gain of chromosome 21. Quantification of ERG DNA copy numbers revealed a strong correlation with ERG mRNA expression. Furthermore, in patients with gain of chromosome 21, higher ERG expression was found to be associated with RUNX1 mutations. Our results suggest that acquired gain of chromosome 21 or amplification of chromosome arm 21q is one mechanism contributing to increased ERG expression in AML.

Genetic characterization of T-PLL reveals two major biologic subgroups and JAK3 mutations as prognostic marker.

T-cell prolymphocytic leukemia (T-PLL) is a rare post-thymic T-cell neoplasm with aggressive clinical course and short overall survival. So far, due to the rareness of this disease, genetic data are available only from individual cases or small cohorts. In our study, we aimed at performing a comprehensive cytogenetic and molecular genetic characterization of T-PLL comprising the largest cohort of patients with T-PLL analyzed so far, including correlations between the respective markers and their impact on prognosis. Genetic abnormalities were found in all 51 cases with T-PLL, most frequently involving the TCRA/D locus (86%). Deletions were detected for ATM (69%) and TP53 (31%), whereas i(8)(q10) was observed in 61% of cases. Mutations in ATM, TP53, JAK1, and JAK3 were detected in 73, 14, 6, and 21% of patients, respectively. Additionally, BCOR mutations were observed for the first time in a lymphoid malignancy (8%). Two distinct genetic subgroups of T-PLL were identified: A large subset (86% of patients) showed abnormalities involving the TCRA/D locus activating the proto-oncogenes TCL1 or MTCP1, while the second group was characterized by a high frequency of TP53 mutations (4/7 cases). Further, analyses of overall survival identified JAK3 mutations as important prognostic marker, showing a significant negative impact.

Amplification of EVI1 on cytogenetically cryptic double minutes as new mechanism for increased expression of EVI1.

In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), increased expression of EVI1 (ecotropic virus integration site 1) was found to be associated with adverse prognosis. Although increased expression of the EVI1 gene is mainly caused by chromosomal rearrangements involving chromosome band 3q26, where EVI1 is located, it can also be identified in cases without these rearrangements. The mechanisms that cause increased EVI1 expression in the absence of 3q26 rearrangements remain unclear. Here, we present four cases with increased EVI1 expression due to EVI1 amplification on cytogenetically cryptic double minutes (dmin). The dmin are small acentric chromosome structures and were observed in about 1% of AML and MDS. We investigated the four cases by conventional cytogenetics, fluorescence in situ hybridization, and array comparative genomic hybridization. Furthermore, EVI1 expression was measured by quantitative reverse transcriptase-PCR. By conventional chromosome analysis, the EVI1 dmin cannot be detected, due to the small size of the amplicons of 0.49-0.78 Mbp. Median % EVI1/ABL expression was 88.9% and therefore comparable to the median % EVI1/ABL expression of patients with EVI1 rearrangements. In conclusion, EVI1 amplification on cytogenetically cryptic dmin causes increased expression of EVI1 and is a new mechanism that causes increased EVI1 expression without a 3q26 rearrangement.

Acute lymphoblastic leukemia with low hypodiploid/near triploid karyotype is a specific clinical entity and exhibits a very high TP53 mutation frequency of 93%.

B lymphoblastic leukemia/lymphoma (ALL) are subdivided by the WHO classification into five subgroups defined by specific translocations and two further subgroups defined by the number of chromosomes. The hypodiploid subgroup is heterogeneous and comprises ALL with a chromosome number of <46. To characterize a specific subset with low hypodiploid karyotype, we performed chromosome banding analysis, FISH, array comparative genomic hybridization, and mutational analyses of FBXW7, NOTCH1, KRAS, NRAS, TP53, and IKZF1 in 29 cases. We observed a nonrandom pattern of chromosome losses, including chromosomes 3, 7, 13, 15, 16, and 17. A deletion encompassing the CDKN2A/B locus was the only recurrent structural abnormality. A duplication of the low hypodiploid karyotype occurred frequently, resulting in a near triploid karyotype based on the definition by merely counting chromosomes but in fact was a very low tetraploid chromosome set. Mutational analyses revealed no mutations in IKZF1, FBXW7, NOTCH1, and KRAS and only one mutation in NRAS. However, we discovered a high frequency of TP53 mutations in 93% (27/29) of cases. In 26/27 cases with TP53 mutation, the second TP53 allele was lost due to monosomy 17. Median overall survival was short (18.5 months), which might be related to the high frequency of TP53 alterations. Therefore, ALL with low hypodiploidy is characterized by a typical pattern of chromosome losses and a remarkably high TP53 mutation frequency. Our data suggest the introduction of a novel WHO entity within the B lymphoblastic leukemia/lymphoma group showing low hypodiploid/very low tetraploid karyotype and concomitant TP53 mutation.

Investigation of 305 patients with myelodysplastic syndromes and 20q deletion for associated cytogenetic and molecular genetic lesions and their prognostic impact.

In patients with myelodysplastic syndromes (MDS), sole 20q deletion [del(20q)] is a recurrent favourable abnormality. We studied additional molecular and cytogenetic lesions and their prognostic impact in 305 MDS patients with del(20q) (229 males/76 females; 29-90 years). All patients were investigated by cytomorphology and chromosome banding analysis (CBA), subsets by fluorescence in situ hybridization, molecular mutation screening, and array comparative genomic hybridization (aCGH). By aCGH (n = 30), the minimal common deleted region (CDR) was flanked by PTPRT (20q13·11) and EYA2 (20q13·12). 210 (68·9%) patients had 'early MDS' without blast increase, 95 (31·1%) 'advanced' MDS with blast increase (5-19%). Additional chromosomal abnormalities (ACAs) were detected in 88/305 (28·9%) patients. Patients with advanced MDS more frequently had ACAs (P = 0·003) and had a higher mean number of ACAs (P = 0·020) and of molecular mutations (P = 0·060). Spliceosome mutations were frequent (U2AF1: n = 31/155; 20·0%; SRSF2: n = 31/159; 19·5%; SF3B1mut: n = 8/159; 5·0%). ASXL1mut (25/153; 16·3%) were associated with advanced MDS (P = 0·001). Presence of ≥3 ACAs (P = 0·003) and ASXL1mut (P = 0·002) were associated with worse 2-year survival. In conclusion, the cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1mut is overrepresented in MDS with del(20q), and ASXL1mut is prognostically adverse.

Three novel cytogenetically cryptic EVI1 rearrangements associated with increased EVI1 expression and poor prognosis identified in 27 acute myeloid leukemia cases.

In acute myeloid leukemia (AML), increased ecotropic virus integration site 1 protein homolog (EVI1) gene expression is prognostically unfavorable. Subsets of cases show 3q26 rearrangements, such as inv(3)(q21q26)/t(3;3)(q21;q26), frequently accompanied by chromosome 7 abnormalities. We investigated whether cytogenetically cryptic EVI1 rearrangements may cause EVI1 overexpression in myeloid malignancies without 3q26 abnormalities and investigated 983 patients with AML (n = 606) or myelodysplastic syndromes (MDS; n = 377) with normal karyotype (CN-AML/CN-MDS, n = 594) or chromosome 7 abnormalities (n = 389) for EVI1 rearrangements using interphase FISH. We identified cytogenetically cryptic EVI1 rearrangements in 27 patients (19 AML, 8 MDS): inv(3)(p24q26) [n = 10]; t(3;21)(q26;q11) [n = 9]; and der(7)t(3;7)(q26;q21) [n = 8]. Elevated EVI1 expression was detected in nearly all cases with cryptic EVI1 rearrangements: Median %EVI1/ABL1 was 92.8 (range: 29.8-146.1) in inv(3)(p24q26), 104.9 (41.4-176.3) in t(3;21)(q26;q11), and 101.8 (4.4-210.4) in der(7)t(3;7)(q26;q21). This was similar to median %EVI1/ABL1 of 73.9 (range: 7.3-585.6) in an independent cohort of inv(3)(q21q26)/t(3;3)(q21;q26) and 67.1 (2.3-410.7) in other 3q26/EVI1 rearrangements. Healthy controls showed median EVI1 expression of 0.5 (range: 0.0-5.8). Using SNP microarray and sequencing analyses, the breakpoints of der(7)t(3;7)(q26;q21) were assigned to CDK6 and centromeric of EVI1, and of t(3;21)(q26;q11) to be within EVI1 and NRIP1. Median overall survival in patients with cryptic EVI1 rearrangements was short, comparable to patients with inv(3)(q21q26)/t(3;3)(q21;q26) or other EVI1 rearrangements. Cryptic EVI1 rearrangements contribute to explain the clinical heterogeneity of CN-AML and are associated with elevated EVI1 expression and an unfavorable prognosis. Screening for cryptic EVI1 rearrangements by FISH may be particularly appropriate in CN-AML with elevated EVI1 expression or in AML/MDS patients with chromosome 7 abnormalities.

ETV6 rearrangements are recurrent in myeloid malignancies and are frequently associated with other genetic events.

ETV6 (TEL) rearrangements are favorable in pediatric acute lymphoblastic leukemia but are less well characterized in myeloid malignancies. We investigated 9,550 patients with myeloid disorders for ETV6 rearrangements by chromosome banding analysis and interphase fluorescence in situ hybridization. ETV6 rearrangements were identified in 51 of 9,550 (0.5%) patients (range, 19.2-85.3 years). Frequencies were in detail: acute myeloid leukemia (AML): 40 of 3,798, 1.1%; myelodysplastic syndromes (MDS): 6 of 3,375, 0.2%; myeloproliferative neoplasms (MPNs): 5 of 1,720, 0.3%; MDS/MPN: 0 of 210; and chronic myelomonocytic leukemia: 0 of 447. Thirty-three different partner bands of ETV6 were identified, and most were recurrent: 3q26 (n = 10), 5q33 (n = 4), 17q11 (n = 3), 22q12 (n = 3), 5q31 (n = 2), and 2q31 (n = 2). Additional chromosomal abnormalities were identified in 29 of 51 (57%) ETV6 rearranged cases. In AML, ETV6 rearrangements were frequently associated with NPM1 (9/39, 23%) and RUNX1 mutations (6/31, 19%). The FAB M0 subtype was more frequent in ETV6 rearranged de novo AML than other AML (P < 0.001); expression of CD7 and CD34 by immunophenotyping was higher in ETV6 rearranged AML compared with other subgroups. Survival of 29 ETV6 rearranged de novo AML was compared with 818 AML from other cytogenetic subgroups. Median overall and event-free survival of ETV6 rearranged cases was similar to the intermediate-risk cohort (26.3 vs. 62.2 months and 14.0 vs. 15.4 months) defined according to Medical Research Council criteria. Our study confirms the variety of ETV6 rearrangements in AML, MDS, and MPNs often in association with other genetic events. Prognosis of ETV6 rearranged de novo AML seems to be intermediate, which should be independently confirmed.

Subclones with the t(9;22)/BCR-ABL1 rearrangement occur in AML and seem to cooperate with distinct genetic alterations.

In AML, cooperation of mutations suppressing differentiation ('class-II-mutations') with 'class-I-mutations' increasing cell proliferation is frequent. In rare cases of myeloid malignancies, the BCR-ABL1 fusion was reported to cooperate as class-I-mutation with class-II-mutations, but most cases had to be classified as blast phase of chronic myeloid leukaemia (CML). We identified five cases of Philadelphia positive subclones in AML occurring in coincidence with other genetic lesions: 1:220 patients with inv(16)/CBFB-MYH11 (0·5%), 2:272 AML cases with t(8;21)/RUNX1-RUNX1T1 (0·7%), 1:1029 NPM1-mutated AML (0·1%), and one patient with s-AML following MDS with a 5q-deletion. Four patients had m-BCR (e1a2) BCR-ABL1 transcripts; one case only had an M-BCR (b3a2) breakpoint. These cases allow some interesting conclusions: The BCR-ABL1 rearrangement apparently can cooperate with the NPM1 mutation similar to other class-I-mutations. The identification of Philadelphia positive subclones in <1% of patients with CBF-leukaemias fits well with previous observations that most CBF-AML are accompanied by activating mutations in genes enhancing proliferation. Since we observed the occurrence of the Philadelphia positive subclones at diagnosis, at relapse, or throughout the disease, the time point of the emergence of Philadelphia subclones seems variable in AML. Clinical research should further concentrate on Philadelphia positive subclones in AML to assess the clinical impact.

Comparison of cytogenetic clonal evolution patterns following allogeneic hematopoietic transplantation versus conventional treatment in patients at relapse of AML.

Relapse of acute myelogenous leukemia has been associated with clonal cytogenetic evolution, but no study focused specifically on relapse after allogeneic hematopoietic stem cell transplantation (HSCT). We compared karyotypes in 160 patients at both diagnosis and relapse either after allo-HSCT (n = 26) or standard chemotherapy (n = 134) using chromosome banding analysis combined with fluorescein in situ hybridization. There were 71 females and 89 males (19.7-80.6 years). At diagnosis, aberrant karyotypes were more frequent in the HSCT than in the chemotherapy cohort (16 of 26; 61.5% versus 63 of 134; 47.0%). This was most obvious in patients with unfavorable cytogenetics (8 of 26; 30.8% versus 19 of 134; 14.2%; P = .032). Differences in the karyotypes between diagnosis and relapse were more frequent in the allo-cohort (14 of 26; 53.8% versus 49 of 134; 36.6%) than in the conventional cohort (n.s.), mainly because of newly emerging cytogenetic alterations. Appearance of ≥ 3 new clonal alterations was more frequent in the allo-cohort (6 of 12; 50.0% with clonal evolution versus 5 of 41; 12.2%, P = .005). The mean number of cytogenetic alterations per patient was increasing from 2.0 at diagnosis to 4.0 at relapse in the allo-cohort, in the conventionally treated patients from 0.9 to 1.3 (both P < .001). Thus, higher frequencies of clonal evolution and increasing cytogenetic complexity were observed in the stem cell recipients probably related to the more unfavorable cytogenetic profiles already depicted at diagnosis.