Browsing by Subject "G-banded slides"

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  • Sammallahti, Heidelinde (Helsingin yliopisto, 2020)
    Since the establishment of pathologic and cytogenetic laboratories, left-over material in the form of G-banded slides and cytogenetic fixed cells, as well as formalin-fixed, paraffin-embedded (FFPE) material, tissue samples, blood and bone marrow have been stored in archives for possible later reference. This material, which potentially contains rare and special cases, has been a welcome source for retrospective studies or e.g. for trying out new methods of analysis. Molecular genetic and molecular cytogenetic techniques such as Southern hybridization, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) have been successfully applied on different kinds of archival specimens. With this study we wanted to explore, both through a literature review and through a practical experiment, the history, present day and future use of such archival material in the light of molecular cytogenetics, including the challenges of DNA extraction, sample degradation, data analysis and interpretation as well as ethical issues. The experimental part had two main objectives, (1) to investigate the use of archived cytogenetic material in the form of G-banded slides and cytogenetic fixed cells for array-based CGH (aCGH), and (2) to explore abnormalities on chromosome 1q in hematologic malignancies. Extra material on the long arm of chromosome 1 is a common recurrent chromosomal abnormality that is present in many classes of hematologic cancers as either primary or secondary aberration. It is the most common structural aberration in multiple myeloma (MM), myeloproliferative disease (MPD) and myelodysplastic syndrome (MDS) and is also a frequent aberration in pediatric acute lymphoblastic leukemia (ALL). It has been associated with increased cell proliferation, disease progression and poor outcome, the mechanisms of which are not fully understood yet. To combine these two aims, we screened the patient database for relevant cases and searched the archive for corresponding samples. The idea was to find cases of hematologic malignancies with extra material on chromosome 1q that were available as cytogenetic slides, fixed cells and frozen bone marrow, find an ideal method of DNA extraction from slides (for other material ready protocols were available), analyze the samples with aCGH and compare the results. We wanted to prove the eligibility of archived cytogenetic material for aCGH analysis and at the same time study rearrangements of 1q in our samples. Starting with 38 patient cases, DNA extraction was performed with 2 different protocols, the latter of which, using a modified version of the Puregene® DNA Purification Kit Protocol for Blood Smears, turned out to be more successful. After having obtained sufficient DNA from several slide samples, we assessed DNA quality with agarose gel electrophoresis. Because slide DNA was too fragmented to be used for aCGH and whole genome amplification (WGA) was not a choice, the experiment was continued with archived fixed cells, bone marrow and archived DNA only. Using a high resolution 60-mer oligonucleotide 44K human CGH microarray platform, we analyzed 15 patient cases that were available both as fixed cell and DNA samples (2 cases), both as fixed cell and bone marrow samples (1 case), both as fixed cell and CPT™ cell samples (1 case), frozen bone marrow (7 cases) and DNA samples (4 cases). The malignancies were pediatric ALL (6 cases), adult ALL (1 case), chronic myeloid leukemia (CML, 2 cases), non-Hodgkin lymphoma (NHL, 1 case), Burkitt lymphoma (BL, 3 cases), Hodgkin's disease (HD, 1 case) and one undefined malignancy. After analysis with CGH Analytics software, we saw that aCGH results of fixed cells compared to results from bone marrow or DNA were almost identical, which confirmed that cytogenetic fixed cells were a reliable source for aCGH analysis. Challenges of working with archived material were witnessed in the form of CG-waves and centralization errors and deviations of the hybridization ratio diagram caused by partially degraded DNA. Concerning aberrations calls, 12 of the 15 cases had detectable aberrations on 1q, which included amplification of the whole 1q arm (1 pediatric ALL, 3 BL cases) and duplications or amplifications of parts of 1q (5 pediatric ALL, 2 CML and 1 undefined malignancy cases) in addition to other aberrations. Common break points were 1q21.2 (2 CML cases), 1q23.2 (1 pediatric ALL and 1 CML case) and 1q32.3 (2 pediatric ALL cases), and we detected 2 large common overlapping areas, at 1q21.2q23.2 and 1q25.3q32.2. The areas were, however, too vast for disease gene screening, spanning several Mb each. We could thus prove and confirm the value of cytogenetic archives for scientific studies. Array CGH with fixed cells was confirmed to work well, also frozen bone marrow and archived DNA were valuable sources. We would suggest further aCGH experiments with cytogenetic slides by applying WGA but would also suggest slide and fixed cell material to be used for Next Generation Sequencing, which has not been reported yet. Regarding aberrations on 1q, further studies to more accurately delimit relevant break points and common overlapping areas are recommended.