Barbara Mucha

The application of conventional cytogenetics, FISH, and RT-PCR to detect genetic changes in 70 children with ALL

We investigated bone marrow cells of 70 acute lymphoblastic leukemia children by conventional cytogenetics (CC), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR) methods. CC and RT-PCR for fusion genes BCR/ABL, MLL/AF4, E2A/PBX1, TEL/AML1 were performed at diagnosis in each patient. FISH was performed to verify the presence of fusion genes and MLL rearrangements and to estimate the percentage of abnormal cells. Karyotypes were obtained in 59 (84%) of 70 cases. Thirty-five (59%) of 59 cases revealed chromosome aberrations. Hyperdiploidy >50 chromosomes was present in nine cases, hyperdiploidy 47–50 chromosomes in six, pseudodiploidy in 15, and hypodiploidy in five. BCR/ABL was present in two cases, PBX1/E2A in two, and TEL/AML1 in 14. MLL/AF4 was not found, but the rearrangements of MLL gene were present in five children. The addition of RT-PCR and FISH to CC was of the utmost importance. One of two Ph translocations and one of two t(1;19) were first revealed by RT-PCR. Moreover, FISH showed the percentage of TEL/AML1(+) cells that turned to be an important prognostic factor. The outcome was the best for the children with hyperdiploidy >50 chromosomes without structural changes. It was also good for those with TEL/AML1 present in ≥80% of cells without chromosome aberrations. The presence of pseudodiploidy correlated with poor outcome. The outcome for patients with t(9;22)–BCR/ABL or 11q23–MLL rearrangement was the worst in study group. The presence of BCR/ABL caused eight times increase of risk of death; MLL rearrangements caused 12 times increase.

Different prognosis of acute myeloid leukemia harboring monosomal karyotype with total or partial monosomies determined by FISH: Retrospective PALG study

A monosomal karyotype (MK) was identified by banding techniques (BT) in acute myeloid leukemia (AML). However, BT may be insufficient to determine the actual loss of a complete chromosome, especially in complex karyotypes. We have investigated the effect of monosomy type, total (MK-t) and partial (MK-p), reevaluated by FISH, on prognosis. We have found that complete remission rate and probability of overall survival at 1 year was higher in MK-p (n=27) than MK-t (n=15) group (40% vs. 15.4%, P=0.19 and 30% vs. 9%, P=0.046, respectively). Our results indicate for the first time that monosomy type influences the prognosis of MK-AML.

Concomitance of monosomal karyotype with at least 5 chromosomal abnormalities is associated with dismal treatment outcome of AML patients with complex karyotype – retrospective analysis of Polish Adult Leukemia Group (PALG)

Abstract Monosomal karyotype (MK) and complex karyotype (CK) are poor prognostic factors in acute myeloid leukemia (AML). A comprehensive analysis of cytogenetic and clinical factors influencing an outcome of AML-CK+  was performed. The impact of cladribine containing induction on treatment results was also evaluated. We analyzed 125 patients with AML-CK+  treated within PALG protocols. MK was found in 75 (60%) individuals. The overall complete remission (CR) rate of 66 intensively treated patients was 62% vs. 28% in CK+ MK− and CK+ MK+  group (p = .01). No difference in CR rate was observed between DA and DAC arms. The overall survival (OS) in intensively treated patients was negatively influenced by MK, karyotype complexity (≥5 abnormalities), and WBC >20 G/L in multivariate analysis. The addition of cladribine to DA regimen improved OS only in MK− but not in MK+  group. In conclusion, concomitance of MK with ≥5 chromosomal abnormalities is associated with dismal treatment outcome in AMK-CK+.

Unusual profiles of pediatric acute lymphoblastic leukemia with MLL gene rearrangement

The most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23), which gives rise to the MLL-AF4 fusion gene [1]. MLL (also known as ALL-I, HTRX or HRX) gene translocations are among the most common chromosomal abnormalities recognized in both B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7 – 10% of acute lymphoblastic leukemia and 5 – 6% of acute myeloid leukemia cases [2]. The MLL gene, which is located at the 11q23, has been cloned and has homology to the Drosophila trithorax gene [3]. MLL is required for the proper maintenance of HOX gene expression during development and hematopoiesis. About 50 different MLL fusion partners have been isolated to date, and while similarities exist between groups of partners, there exists no unifying property shared by all the partners. MLL gene rearrangements are found in leukemias with both lymphoid and myeloid phenotypes, and are often associated with infant and secondary leukemias. The immature phenotype of the leukemic blasts suggests an important role, including therapyrelated leukemias, for MLL in the early stages of hematopoietic development [4 – 6]. Rearrangements of chromosome band 11q23 are common in infant leukemias, comprising more than 70% of the observed chromosome abnormalities in children less than 1 year of age [3]. Mixed lineage leukemia (MLL) abnormalities occur in *50% of childhood pre-pre-B acute lymphoblastic leukemia [7]. In spite of the fact that it is one of the most common nonrandom chromosomal abnormalities, there are scant data showing occurrence of MLL rearrangement in childhood ALL with different phenotype, such as common-ALL or T-ALL. We analyzed 121 consecutive pediatric ALL cases diagnosed and treated in one center. All children were screened at diagnosis for immunophenotype, DNA index, karyotype by G-banding method, MLL, and BCR-ABL gene rearrangements by FISH (fluorescence in situ hybridization) and RT-PCR. Both conventional cytogenetics and FISH were carried out. FISH analysis was performed utilizing commercially available DNA probes, including whole chromosome painting probes, locus specific probes, and specific dual color translocation fusion probes. As far as MLL rearrangements are concerned, FISH can not only detect reciprocal translocation but also a deletion of at least 190 kb from the 30 region of the MLL gene [8]. Common/ pre-B-ALL immunophenotype was diagnosed as CD19þ, HLA-DRþ, CD10þ, and surface immunoglobulin7 [sIg7]; pre-pre-B-ALL (pro-B-ALL) immunophenotype as (CD19þ, HLA-DRþ, CD107, cytoplasmic m chain7 and surface immunoglobulin7 [sIg7]). Mature B-ALL cells characterized

CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis

Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.

Hiperdiploidia limfoblastów i jej związek z immunofenotypem w ostrej białaczce limfoblastycznej

Wstep Do najwazniejszych badan diagnostycznych w ostrej bialaczce limfoblastycznej (ALL) zalicza sie: badania morfologiczne, badania cytochemiczne, immunofenotypowanie, badania indeksu DNA i analize cyklu komorkowego oraz badania cytogenetyczne i molekularne aberracji chromosomowych. Cel pracy Analiza związku wynikow badania immunologicznego i analizy faz cyklu komorkowego określanych metodą cytometrii przeplywowej z obrazem cytogenetycznym przy rozpoznaniu ostrej bialaczki limfoblastycznej u dzieci. Pacjenci i metodyka Analize profilu diagnostycznego pacjentow z nowo rozpoznaną ALL przeprowadzono u 151 dzieci. Za kryterium rozpoznania ostrej bialaczki limfoblastycznej przyjeto stwierdzenie w szpiku kostnym co najmniej 20% blastow bialaczkowych. Immunofenotyp blastow i indeks DNA oznaczano metodą cytometrii przeplywowej. Badania cytogenetyczne wykonano metodą prązkową, a badania molekularne metodami FISH i PCR. Wyniki i wnioski Pacjenci z ostrą bialaczką limfoblastyczną z wartością indeksu DNA limfoblastow powyzej 1,16 mają immunofenotyp charakterystyczny dla prekursorow limfocyta B, w przewazającej wiekszości przypadkow z obecnością antygenu common CD10. Aberracje cytogenetyczne limfoblastow o charakterze hiperdiploidii lub rearanzacji genow TEL-AML1 stwierdzono wylącznie wśrod pacjentow z immunofenotypem common-ALL. Nie stwierdzono znaczenia prognostycznego wartości indeksu DNA limfoblastow. Korzystny profil cytogenetyczny wyrazający sie hiperdiploidią limfoblastow lub obecnością translokacji t(12;21) lub rearanzacji TEL-AML1 ma bardzo korzystne znaczenie rokownicze w ostrej bialaczce limfoblastycznej.

Primary myelofibrosis with normal karyotype and cryptic aberrations detected by FISH: case report

Introduction. Myeloproliferative neoplasms (MPNs) are the result of clonal haematopoietic stem cell disorders. The most common cytogenetic aberrations are: partial trisomy 1q, 13q–, 20q–, trisomy 8 and abnormalities of chromosomes 1, 7 and 9. Conventional karyotyping is a routinely used method. Fluorescent in situ hybridisation (FISH) analysis however may also be an integral component of the diagnostic evaluation, especially when the abnormality is cryptic. Subject and methods. A 70-year-old woman was admitted to the Department of Haematology in September 2013 with suspected acute myeloid leukemia (AML). The final diagnosis was primary myelofibrosis and NYHA class III heart failure. Bone marrow (BM) was used for karyotyping and FISH. Peripheral blood (PB) was used for PCR. Results. Cytogenetic GTG analysis revealed normal female karyotype — 46,XX [22]. The result of analysis of JAK2 V617F mutation was negative. Analysis using LSI BCR/ABL Dual Fusion Probe, JAK2 Break Probe and RB1 Deletion Probe showed abnormal cells, of which the numbers were beyond the normal cutoffs. FISH examinations using p53 Deletion Probe and LSI CDKN2A/CEP 9 showed normal cells. Conclusion. The diagnosis of primary myelofibrosis may pose a problem. We still do not know the specific abnormalities (i.e. genomic and chromosomal aberrations or gene mutations), the occurrence of which may help to diagnose and assess a probable time of survival of patients with PMF. Further examinations are needed (e.g. using aCGH) to find out more about myeloproliferative neoplasms.

Profil diagnostyczny wznowy ostrej białaczki limfoblastycznej

Ostra bialaczka limfoblastyczna (ALL) jest najczestszym nowotworem u dzieci. Pomimo stalego postepu w terapii ALL, u okolo 20% pacjentow z tą chorobą dochodzi do wznowy. Szacuje sie, ze wznowa ostrej bialaczki limfoblastycznej moze byc traktowana jako czwarty co do czestości nowotwor wieku dzieciecego. Cel pracy Analiza profilu diagnostycznego ALL w okresie rozpoznania i we wznowie szpikowej. Pacjenci i metodyka Badaniom poddano 24 pacjentow ze wznową szpikową ALL, u ktorych przeprowadzono analize profilu diagnostycznego choroby w momencie rozpoznania i jej wznowy (morfologia limfoblastow wg klasyfikacji FAB, badania cytochemiczne, analiza immunofenotypu metodą cytometrii przeplywowej, ocena indeksu DNA i analiza cyklu komorkowego, analiza wynikow badan cytogenetycznych i molekularnych). Wyniki We wznowie stwierdzono zmiany w morfologii blastow (u 3 pacjentow z L1 na L2), zmiany reakcji PAS lącznie u 4 dzieci, zmiany w immunofenotypie u 7 pacjentow, w tym u 4 z ALL z linii B-komorkowej oraz u 3 z rozpoznaniem ALL linii T-komorkowej. Zmiana immunofenotypu byla związana ze zmianą wartości indeksu DNA w 3 przypadkach. Stwierdzono 2,4-krotnie wieksze ryzyko wznowy u pacjentow, u ktorych na limfoblastach nie wystepowal antygen CD10 (tj. pre-pre-B-ALL lub T-ALL) niz u pacjentow z obecnością CD10 (tj. common-pre-B-ALL). We wznowie stwierdzono nieprawidlowy kariotyp limfoblastow u wiekszości pacjentow. Wniosek We wznowie ALL u dzieci stwierdza sie zmiany w morfologii i immunofenotypie limfoblastow, czestsze wystepowanie bardziej niedojrzalego immunofenotypu, zlozonych zaburzen cytogenetycznych oraz rzadkie wystepowanie korzystnych zmian cytogenetycznych.