Katarzyna Skonieczka

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.

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+.

Cytogenetics in Hematology

According to European LeukemiaNet—Workpackage Cytogenetics, cytogenetic analysis is mandatory to correctly diagnose and classify hematologic malignancies. Conventional cytogenetic analysis, which enables detection of both balanced and unbalanced chromosomal rearrangements, is still regarded as the gold standard of genetic diagnostics in hemato-oncology. However, a variety of rapidly developing state-of-the-art molecular and cyto-molecular techniques can now be applied, complementing the process of detection and analysis of recurrent genetic aberrations. Cytogenetics is currently recognized as an essential part of diagnostic process in hematologic malignancies. It helps to define specific disease entities, provides important prognostic and predictive information, influences therapy by setting the basis for individual treatment options that target cancer specific genetic abnormalities or their products, as well as helps to assess therapy effectiveness by indicating genetic remission or progression.

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.

Constitutional mutations of the CHEK2 gene are a risk factor for MDS, but not for de novo AML

CHEK2 plays a key role in cellular response to DNA damage, and also in regulation of mitosis and maintenance of chromosomal stability. In patients newly diagnosed with myelodysplastic syndrome (MDS, n = 107) or acute myeloid leukemia (AML, n = 117) congenital CHEK2 mutations (c.444 + 1G > A, c.1100delC, del5395, p.I157 T) were tested by PCR and sequencing analysis. The karyotype of bone marrow cells of each patient was assessed at disease diagnosis using classical cytogenetic methods and fluorescence in situ hybridization. The CHEK2 mutations were strongly associated with the risk of MDS (p < 0.0001) but not with the risk of de novo AML (p = 0.798). In CHEK2-positive MDS patients, two times higher frequency of aberrant karyotypes than in CHEK2-negative patients was found (71% vs. 37%, p = 0.015). In CHEK2-positive patients with cytogenetic abnormalities, subtypes of MDS: refractory anemia with excess blasts-1 or 2, associated with unfavorable disease prognosis, were diagnosed two times more often than in CHEK2-negative cases with aberrations (78% vs. 44%). In conclusion, the congenital CHEK2 inactivation is strongly associated with the risk of MDS and with a poorer prognosis of the disease. However, the chromosomal instability in AML is not correlated with the hereditary dysfunction of CHEK2.

Prognostic significance of BCR-ABL rearrangement in childhood acute lymphoblastic leukemia

B a c k g r o u n d . Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy. Presence of adverse risk factors determines risk group stratification in this disease. O b j e c t i v e . The aim of study was the analysis of results of therapy and role of prognostic risk factors in treatment of childhood ALL in kujawsko-pomorskie region in 1995-2010. P a t i e n t s a n d m e t h o d s . During this period, ALL was diagnosed in 223 patients. With respect to time period and therapy protocol, the patients were divided into two groups: group 1 A/B (1995-2002) and group 2 (2002- 2010). Probability of overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) were analyzed. Uni- and multivariate analyzes for risk factors were performed. R e s u l t s . Over the analyzed 17-year period, OS has increased from 77.9% in group 1A and 73.7% in group 1B to 86.2% in group 2. Results of RFS and EFS have also increased during this time. The death rate has decreased from 26% in group 1A and 26.3% in group 1B to 10.2% in group 2. The most important adverse prognostic risk factors during the first period included involvement of liver, spleen, lymph nodes as well as poor response to initial therapy, while during the second period the most important independent risk factor was BCR-ABL rearrangement in lymphoblasts. C o n c l u s i o n s . The most important independent prognostic risk factors in pediatric ALL include advanced disease, BCR-ABL rearrangement, and initial response to therapy. These factors are used for stratification to treatment groups, intensification of therapy and hematopoietic stem cell transplantation.

The hematological malignancies related to primary hypereosinophilia and their diagnostics

Published in 2008, by experts of the World Health Organization, the new classification of hematological malignancies forced a change of look at chromosomal aberrations and gene mutations, which are important in establishing the diagnosis and prognosis for patients with these malignancies. The new classification includes a new category of neoplasms - hematological malignancies with hypereosinophilia. Due to the high diversity of causes of hypereosinophilia and underlying genetic changes, their differential diagnosis is based on classical cytogenetics, fluorescence in situ hybridization (FISH) and genetic molecular techniques. Cytogenetic analysis of bone marrow cells showed that the majority of hypereosinophilia cases can be characterized by the presence of normal karyotype. Therefore, routine cytogenetic diagnostics should be complemented by FISH with break-apart probes for potentially rearranged genes (e.g., CBFB, ETV6) and unique probes for fusion genes (e.g., FIP1L1-PDGFRA), specific for hypereosinophilia-associated diseases. In differential diagnosis of hypereosinophilia, the analysis of characteristic gene mutations (e.g., cKIT) and gene fusions (e.g., ETV6-PDGFRB) is also applied, using molecular genetic methods.

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.