The use of interphase fluorescence in situ hybridization for cytogenetic analysis on paraffin sections in children with non-Hodgkin lymphomas

Wstęp. Według klasyfikacji WHO wykrycie aberracji cytogenetycznych w komórkach nowotworowych pozwala nabardziej precyzyjne określenie podtypu chłoniaka. Technika FISH daje możliwość analizy zmian genetycznych w komórkachznajdujących się w różnych fazach cyklu komórkowego.Celem pracy było opracowanie metody diagnostyki zmian cytogenetycznych z zastosowaniem techniki FISH do jąderinterfazowych, uzyskiwanych z bloczków parafinowych, w przypadkach nieziarniczych chłoniaków złośliwych u dzieci.Materiał i metody. Badania przeprowadzono u 38 chorych z chłoniakami nieziarniczymi, w wieku od 1–16 lat(mediana 8,3) zdiagnozowanych w latach 1993–2000. Przedmiotem badań były fragmenty tkanki nowotworowejzatopione w parafinie. W I etapie pracy doświadczalnie opracowano metodę ekstrakcji jąder komórkowych zeskrawków parafinowych. W II etapie zastosowano procedurę FISH do jąder interfazowych uzyskanych z bloczkówparafinowych od 28 dzieci.Wyniki. Doświadczalnie ustalono optymalne parametry izolacji jąder komórkowych ze skrawków parafinowych.Po zastosowania techniki FISH do jąder interfazowych uzyskano pozytywny wynik hybrydyzacji w 36/40 badanychpreparatów (90%), od 24/28 dzieci (87,5%); aberracje chromosomowe stwierdzono u 11/28 chorych (39,3%), w tym:w 6/17 przypadków chłoniaka B-komórkowego, w 2/5 limfoblastycznego i 3/6 anaplastycznego wielkokomórkowego.Wnioski. Analiza FISH na materiale uzyskanym z bloczków parafinowych niesie za sobą trudności interpretacyjne.Zastosowanie metody izolacji jąder komórkowych zwiększa wykrywalność zmian cytogenetycznych w archiwalnymmateriale biopsyjnym techniką FISH. Warunkiem wykorzystania materiału archiwalnego do analizy FISH jest standaryzacjametod utrwalania materiału w parafinie. Ocena znaczenia wykrytych zmian cytogenetycznych wymagadalszych badań z dużą grupą pacjentów poddanych długotrwałej obserwacji.Background. According to WHO classification, the detection of genetic alterations in tumor cells enables definition ofthe subtype of lymphoma more precisely. The FISH technique allows analysis of cytogenetic changes in the cells in anyphase of the cell cycle. The aim of this work was to develop the method of cytogenetic analysis with the use of FISHon interphase nuclei extracted from paraffin-embedded sections, in cases of children with non-Hodgkin lymphomas.Material and methods. The study population was 38 children with non-Hodgkin lymphoma aged from 1 to 16 years(median age 8.3), diagnosed between 1993 and 2000. We tested paraffin-embedded malignant tissues. In the first stage of the work we developed the method of nuclei extraction from paraffin sections, then in the second stage we used it with FISH analysis in material from 28 children.Results. We devised the optimal patterns of nuclei extraction from paraffin sections. FISH performed on the extracted interphase nuclei were positive in 36/40 (90%) of tested specimens of 24/28 patients (85.7%). We detected the cytogenetic changes in 11/28 patients (39.3%): in 6/17 cases of B-cell lymphoma, 2/5 lymphoblastic lymphoma and 3/6 anaplastic large cell lymphoma.Conclusions. FISH analysis performed on paraffin-embedded tissues is, due to different reasons, difficult to evaluate. The introduction of interphase nuclei extraction method allows the increase in the detection of cytogenetic alterations by FISH in archival biopsed material. However, this technique requires an efficient fixation procedure. To define the significance of cytogenetic aberrations in pediatric lymphomas, further studies of large series of patients with long follow-up should be undertaken

Outcome of refractory and relapsed acute myeloid leukemia in children treated during 2005-2011 : experience of the Polish Pediatric Leukemia/Lymphoma Study Group (PPLLSG)

AIM OF THE STUDY: Recent studies showed relatively better outcome for children with refractory (refAML) and relapsed acute myeloid leukemia (relAML). Treatment of these patients has not been unified within Polish Pediatric Leukemia/Lymphoma Study Group (PPLLSG) so far. The goal of this study is to analyze the results of this therapy performed between 2005–2011. MATERIAL AND METHODS: The outcome data of 16 patients with refAML and 62 with relAML were analyzed retrospectively. Reinduction was usually based on idarubicine, fludarabine and cytarabine with allogenic hematopoietic stem cell transplant (alloHSCT) in 5 refAML and 30 relAML children. RESULTS: Seventy seven percent relAML patients entered second complete remission (CR2). Five-year OS and disease-free survival (DFS) were estimated at 16% and 30%. The outcome for patients after alloHSCT in CR2 (63%) was better than that of those not transplanted (36%) with 5-year OS of 34% vs. 2-year of 7% and 5-year DFS of 40% vs. 12.5%. Second complete remission achievement and alloHSCT were the most significant predictors of better prognosis (p = 0.000 and p = 0.024). The outcome of refAML children was significantly worse than relAML with first remission (CR1) rate of 33%, OS and DFS of 25% at 3 years and 53% at 2 years, respectively. All survivors of refAML were treated with alloHSCT after CR1. CONCLUSIONS: The uniform reinduction regimen of the documented efficacy and subsequent alloHSCT in remission is needed to improve the outcome for ref/relAML children treated within PPLLSG. The focus should be on the future risk-directed both front and second line AML therapy