Standard Cytogenetic Analysis Research Articles

Association of WNT Gene Polymorphism with Frequency of Cytogenetic Disorders under the Action of Ionizing Radiation

The paper presents the results of a study of the association of single nucleotide polymorphisms of the WNT genes with an increased frequency of cytogenetic disorders in the blood lymphocytes of workers at an ionizing radiation facility exposed to long-term radiation exposure at doses of 100–500 mGy.The object of the study was the blood of 95 apparently healthy workers who were subjected to long-term technogenic external exposure to γ-radiation in doses from 100 to 500 mGy in the course of their professional activities. For all examined individuals, a standard cytogenetic analysis of blood lymphocytes was performed. Genomic DNA was isolated from workers’ blood lymphocytes using a “QIAamp DNA Blood mini Kit” (Qiagen, Germany). DNA was genotyped for 116 single nucleotide polymorphisms of the WNT genes using high-density “CytoScan™ HD Array” (Affymetrix, USA) chips (DNA chips). Taking into account the Bonferroni correction, an association of single nucleotide polymorphisms of the WNT genes with a high frequency of circular chromosomes in blood lymphocytes was established, all other types of cytogenetic disorders did not show statistical significance. As a result of the study, a single nucleotide polymorphism of the WNT9B gene rs1530364 was identified, which can be considered as a potential marker of individual radiosensitivity.

Contribution of chromosomal aberrations to the pathogenesis of primary and secondary amenorrhea: A study from Western Iran.

Amenorrhea is an abnormal condition characterized by the absence of menstruation in women of reproductive age. According to the World Health Organization, amenorrhea ranks as the sixth leading cause of female infertility. Approximately 2% to 5% of women of reproductive age experience amenorrhea, which can be classified as primary amenorrhea (PA) or secondary amenorrhea (SA). Several studies have named chromosomal abnormalities among the main causes of amenorrhea, though the prevalence of these abnormalities may differ across populations. The objective of this study was to ascertain the frequency and types of chromosomal abnormalities in women with amenorrhea in Kermanshah Province, Iran. This retrospective study included patients with PA and SA who underwent standard cytogenetic analysis. We also conducted a review of the literature on chromosomal abnormalities and their prevalence in SA. Among the 137 cases of PA in this study, 22% exhibited chromosomal abnormalities. Numerical changes were the most common finding (46.6%) in this group, including 45,X, mosaic, and 47,XXX karyotypes. These were followed by the 46,XY karyotype (40%). Of the 51 cases of SA that received chromosomal analysis, abnormalities were identified in only one case. Additionally, our review of the literature revealed that chromosomal aberrations are responsible for 7% of SA cases globally. In this study, we successfully characterized the cytogenetic causes of PA and SA in a substantial population from Kermanshah Province, Iran.

Interphase nuclei, karyotypes and nuclear DNA amounts in five species of Oenocarpus (Arecaceae).

The genus Oenocarpus Martius, 1823 (Arecaceae) includes five species commonly used in Amazonia, especially for their fruits. Little is known about the cytogenetic characteristics and DNA amounts of these species, except for O.bataua (Martius, 1823). This study characterized and compared the types of interphase nuclei, the chromosome sets, and estimated the nuclear DNA amounts of Oenocarpusbacaba (Martius, 1823), O.bataua, O.distichus (Martius, 1823), O.mapora (H. Karsten, 1857) and O.minor (Martius, 1823). Standard cytogenetic analyses and estimates of the nuclear DNA amount by flow cytometry were carried out. These are the first reports of chromosome numbers and DNA amounts, except for O.bataua, as is the description of the chromatin distribution in interphase nuclei and karyotype for all species. All species presented 2n = 36, confirming the previous report for O.bataua. Differences between karyotype formulas and the positioning of secondary constrictions were observed. There were no significant differences for the nuclear DNA amounts among species. The constancy in chromosome number and variations in karyotype formulas suggest the occurrence of chromosome rearrangement as an important mechanism in Oenocarpus speciation.

Фолликулярная лимфома 1–3А цитологического типа с наличием или отсутствием t(14;18)(q32;q21): прогноз, выбор противоопухолевой терапии и ее результаты

Aim. To determine the prognostic value of t(14;18)(q32;q21) in follicular lymphoma (FL) of grades 1–3А, to assess the chemotherapy efficacy in “t(14;18)+ FL” and “t(14;18)– FL” patients, and to analyze the cases of ineffective therapy.
 Materials & Methods. The retrospective/prospective study carried out at the National Research Center for Hematology in the period of 2001–2022 enrolled 362 patients with newly diagnosed FL of grades 1–3А. Their risk stratification was based on predictive models FLIPI and PPI3 (Personalized Predictive Index[1]). The patients were 30–81 years of age (median 52 years). There were 225 women and 137 men. They received the following regimens: R-B (n = 80), R-CHOP (n = 189), R-CHOP (4 cycles) + R-DHAP (2 cycles) (n = 28), and R-CHOP (4 cycles) + R-DHAP (2 cycles) + auto-HSCT in the first-line therapy (n = 65). For 2 years, maintenance rituximab therapy was administered to all the enrolled patients, whichever drug chemotherapy they received. Standard cytogenetic analysis and FISH were carried out in 265/362 (73 %) patients.
 Results. Patients were divided into two comparable groups: “t(14;18)+ FL” (n = 196) and “t(14;18)– FL” (n = 69). Patients without cytogenetics/FISH (n = 97) were excluded from the analysis. In patients without t(14;18), poor prognostic factors, such as grade 3А (p = 0.003) and Ki-67 > 35 % (p = 0.001), were identified significantly more often, and also high PPI3 risk was reported (p = 0.008). No differences (p = 0.84) were detected during FLIPI risk stratification of patients. Bone marrow lesions were observed significantly more often in “t(14;18)+ FL” compared to “t(14;18)– FL” (p = 0.002). The chemotherapy outcomes, such as 2-year EFS and OS, appeared to be considerably worse in “t(14;18)– FL” compared to “t(14;18)+ FL” patients.
 Conclusion. The group of FL patients with t(14;18) appeared to be most numerous and more prognostically favorable. Immunochemotherapy regimens R-B and R-CHOP are more justified in the first-line therapy of FL with low PPI3 risk. Therapy outcomes were comparable in efficacy. In intermediate and high PPI3 risk FL patients with t(14;18), the most effective first-line therapy was the one with consistent administration of R-CHOP, R-DHAP, and auto-HSCT. Based on the results of this study, FL of grades 1–3А without t(14;18) can well be considered to be a prognostically unfavorable variant of this malignant lymphoid tumor. The rate of early relapses/progression after the standard immunochemotherapy (R-B and R-CHOP), according to our data, is 60 %. In patients with newly diagnosed FL who received consistent administration of R-CHOP, R-DHAP, and auto-HSCT in the first-line therapy, this rate drops to 30 %. Our results clearly indicate the need for new FL treatment approaches.

Genome sequencing identify chromosome 9 inversions disrupting ENG in 2 unrelated HHT families

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is a dominant inherited vascular disorder. The clinical diagnosis is based on the Curaçao criteria and pathogenic variants in the ENG and ACVRL1 genes are responsible for most cases of HHT.Four families with a negative targeted gene panel and selected by a multidisciplinary team were selected and whole-genome sequencing was performed according to the recommendations of the French National Plan for Genomic Medicine. Structural variations were confirmed by standard molecular cytogenetic analysis (FISH).In two families with a definite diagnosis of HHT, we identified two different paracentric inversions of chromosome 9, both disrupting the ENG gene. These inversions are considered as pathogenic and causative for the HHT phenotype of the patients.This is the first time structural variations are reported to cause HHT. As such balanced events are often missed by exon-based sequencing (panel, exome), structural variations may be an under-recognized cause of HHT. Genome sequencing for the detection of these events could be suggested for patients with a definite diagnosis of HHT and in whom no causative pathogenic variant was identified.

СРАВНИТЕЛЬНЫЙ РЕТРОСПЕКТИВНЫЙ АНАЛИЗ РЕЗУЛЬТАТОВ ЦИТОГЕНЕТИЧЕСКИХ ИССЛЕДОВАНИЙ РАБОТНИКОВ ОБЪЕКТА ИСПОЛЬЗОВАНИЯ ИОНИЗИРУЮЩЕГО ИЗЛУЧЕНИЯ

Purpose: To conduct a comparative retrospective analysis of the results of cytogenetic studies of workers at facilities using ionizing radiation in 2003 and 2018. Material and methods: The material for the study was the venous blood of workers at facilities using ionizing radiation. The study was conducted on conditionally healthy workers (n = 11), of which 2 groups were formed: the control group (blood sampling and cytogenetic study of blood lymphocytes were carried out in 2003) and the study group (blood sampling was carried out in 2018). For all examined individuals, blood lymphocytes were cultured and standard cytogenetic analysis followed by statistical processing of the results. Results: A comparative retrospective analysis showed that in the study group (2018) compared to the control group (2003), the frequency of chromatid fragments was reduced (p = 0.0452). The frequencies of other types of cytogenetic disorders studied (aberrant cells, chromosomal fragments, dicentric and circular chromosomes) do not differ between groups. Cytogenetic abnormalities in blood lymphocytes are a highly sensitive measure of the degree of radiation exposure in the early and late periods after exposure and can be used as a biological indicator of ionizing radiation. The absence of differences in indicators of radiation exposure markers may be due to the large interval of time that elapsed after irradiation until the moment of examination (15 years), during which lymphocytes with chromosomal aberrations were eliminated from the blood. In the future, for the completeness of the retrospective analysis and the accuracy of the result, it is planned to conduct a study on a larger sample with a shorter time interval between cytogenetic studies. Conclusion: The result of this work allows us to supplement the understanding of the mutation process in the somatic cells of individuals exposed to ionizing radiation in the course of professional activities and indicates genotoxicity.

Detection of Cryptic Gene Fusion and Chimeric RNA Variants in Relapsed/Refractory Acute Myeloid Leukemia Patients Diagnosed with KMT2A/Afdn Chromosomal Translocation

Chromosomal rearrangements, such as translocations, play a significant role in creating chimeric genes and oncofusion proteins initiating leukemogenesis in 52% of acute myeloid leukemia (AML) patients, and are associated with an adverse prognosis. Currently, there are limited targeted therapies available for AML patients with chromosome rearrangements primarily due to a lack of accurate molecular and genetic characterization of the mechanism of leukemic progression along the treatment course. In our study, we investigated the pathological landscape of treatment resistance and relapse in AML patients with KMT2A-rearranged gene fusions (previously known as mixed lineage leukemia, MLL gene, 11q23), which is one of the most common gene rearrangements in AML with a dismal prognosis. Our approach included chromosome analysis, fluorescence in situ hybridization (FISH), transcriptomic analyses (RNA-seq), polymerase chain reaction (PCR), DNA-sequencing and quantitative PCR (qPCR) were applied to monitor RNA- and genome-wide changes among newly diagnosed, refractory and relapsed AML patients. Bone marrow samples of seven AML patients with different chromosome were obtained and two samples were found to possess the chromosomal translocation t(6;11)(q27;q23) which leads to KMT2A/AFDN gene fusion. In these patients, RNA-seq identified a cryptic CCDC32 (15q15.1) /CBX3 (7p15.2) gene fusion in both newly diagnosed and relapsed samples, which is previously unreported in KMT2A/AFDN-rearranged AML patients. Quantitative gene expression measurement by qPCR demonstrated that CCDC32/CBX3 gene fusion significantly affected the expression of wild-type genes of CCDC32 (essential for embryonic development) and CBX3 (an oncogene for solid tumors) during the relapse, suggesting the potential oncogenic role for the CCDC32/CBX3 chimera in AML development. Our result is consistent with a previous study showing multiple breakpoints in AML patients with KMT2A/AFDN translocations. KMT2A rearrangements are known to have an unfavorable prognosis in AML and the specific t(6;11)(q27;23) translocation in AML patients leads to a median overall survival of only 12 months. Our novel findings also suggest that one reason for the adverse outcomes of KMT2A chromosomal rearrangements is their complex ability to generate chimeric variants and more aggressive subclones to elude the treatment. Our study implies that standard cytogenetic analyses and emerging NGS are not sufficient to fully diagnose a patient with chromosome rearrangement. Particularly, short-read RNA-seq presents problems because it cannot detect gene fusions with low expression level, like the KMT2A/AFDN gene fusion that was undetected in patient 7 by our short-read RNA-seq. Instead, long-read RNA-seq, combined with cytogenetics and molecular profiling through PCR/Sanger DNA-sequencing, should be utilized to identify complex fusion transcripts and different chimeric variants of multiple gene fusions.

Search for polymorphic variants of candidate genes contributing to individual radiosensitivity

Background.Cytogenetic damage (СD) in lymphocytes induced by low doses (up to 0.1 Sv) of ionizing radiation (IR) is the main cytogenetic sign of individual radiosensitivity of the human body. In addition to DNA repair and cell death, which affect the formation of СD and its elimination, IR effects on the cell can be manifested through changes in proliferation of cells with unrepaired DNA damage. The system of cyclins and cyclin-dependent kinases (CDK), which provide coordination of mitotic events during passage of a cell through the cell cycle, plays a crucial role in regulation of cell proliferation.Aim.To evaluate the relationship of single-nucleotide polymorphisms (SNPs) of cell cycle genes with an increased frequency of СD in workers of a nuclear power plant affected by chronic occupational radiation exposure in the dose range of 100–500 mSv.Materials and methods.The object of the study was blood of 55 conditionally healthy workers of Siberian Chemical Plant (SCP) who were affected by chronic occupational radiation exposure (gamma radiation) in the dose range of 100–500 mSv. A standard cytogenetic analysis of blood lymphocytes was performed for all examined individuals. Genomic DNA was isolated from the blood of the workers using the QIAamp DNA Blood Mini Kit (QIAGEN, Germany). DNA was genotyped using 257 SNPs of cyclin genes and neighboring intergenic regions using DNA microarrays from the high-density CytoScan HD Array (Affymetrix, USA).Results.Taking into account the Bonferroni correction, only statistically significant associations of SNPs with the frequency of dicentric chromosomes were found; all other types of chromosomal aberrations did not show statistical significance. The rs803054CCNI2was associated with an increased frequency of dicentric chromosomes arising under the influence of chronic occupational radiation exposure.Conclusion.The discovered SNP (rs803054), whose recessive genotype is associated with an increased frequency of dicentric chromosomes in workers of SCP exposed to radiation at doses of 100–500 mSv over a long time, can be considered as a potential marker of individual radiosensitivity. To confirm the identified associations, further validation studies are needed on an expanded sample of people affected by chronic occupational radiation exposure.

Accurate Detection of Clinically Actionable Copy Number Variants in Diverse Hematological Neoplasms By Routine Targeted Sequencing: A Comparative Performance Study

Background: Accurate genotyping of Copy Number Variations (CNVs) is an essential part of the clinical evaluation of diverse hematologic malignancies. Conventional karyotyping and FISH are current gold standards to assess CNVs, but both have some limitations. Next-generation targeted sequencing panels are commonly incorporated into current clinical practice in most cancer centers. We examined the utility of CANARy (Copy number ANomaly Assessment and Recovery), a CNV profiling method for three hematological malignancies using targeted panel sequencing data. Patients and Methods: We studied patients with myelodysplastic syndrome (MDS), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and plasma cell disorders (PCD) identified from a cohort of 4752 hematological specimens profiled at Stanford University by conventional cytogenetics and next-generation sequencing (NGS) between 2018 and 2021. We curated a cohort of 200 patients diagnosed with MDS (n=72), CLL (n=46), and PCDs including MM, AL amyloidosis, and MGUS (PCD, n=82), along with 50 cytogenetically normal controls. Specimens were bone marrow aspirates, peripheral blood, or FFPE tissue. All samples were characterized by conventional metaphase karyotyping and/or FISH and we focused on the analysis of clinically relevant CNV (-5q, -7/-7q, -20q, +8 for MDS; -11q22, -13q14, -17p13, +12 for CLL; and +1q21, -1p32, -13q14, -17p.13 for PCD). The specimens separately had clinical NGS (164 genes, Heme-STAMP) performed no more than four weeks from standard cytogenetic analysis. We applied multiple CNV enumeration tools to sequencing data for CNV genotyping at these loci, including our recently developed method CANARy (Chabon et al, Nature 2020). CANARy is a read depth-based, genome-wide CNV caller from targeted sequencing data that combines information from on-target and off-target fragments to assign a final Z-score and copy number index to a set of pre-defined genomic regions. We benchmarked CANARy alongside three others publicly available CNV callers: CNVkit (Talevich et al, PLoS Comput Biol. 2016), ichorCNA (Adalsteinsson et al, Nat Commun. 2017), and WISECONDOR (Straver et al, Nucleic Acids Res. 2014). All tools were run using the default parameters and following author recommendations. We calculated the accuracy and other statistics parameters for each CNV caller, considering karyotype/FISH as the true reference. We estimated tumor fraction using two strategies: the variant allele fraction (VAF) of disease-related driver mutations and using the ichorCNA estimate. Results: A total of 211 CNVs were identified by karyotype and/or FISH, with disease-specific distribution summarized in Fig. A. CANARy detected 148/211 (70.1%), CNVkit 88/211 (41.7%), ichorCNA 116/211 (55%) and WISECONDOR 158/211 (74.9%). The overall accuracy for CANARy, CNVkit, ichorCNA and WISECONDOR was 86.3%, 81.8%, 84.3% and 83.1% respectively. CANARy had both the highest overall accuracy (86.3%) and overall F1-score (0.73) (Fig.B). WISECONDOR had the highest number of false positives (83 vs. CANARy = 48). Interestingly, all CNV tools performed better in MDS specimens, with 92% accuracy for CANARy in MDS samples, likely related to the higher neoplastic purity of these samples. The median ichorCNA tumor fraction estimates for MDS, CLL and PCD were 22% (0-99%), 14% (0-70%), and 7% (0-94%), respectively. In addition, CNV calling was inferior in PCD tumors than other tumor types. Within CLL, -11q22 and -13q14 lesions were more challenging to detect with any of the four methods, with CANARy sensitivity of 50% and 31.3% respectively, likely related to the smaller minimal deletion regions of these lesions. The 17p deletion in CLL cases, known for its poor prognostic impact, was best identified by CANARy; with a 100% sensitivity and a 97.6% specificity. The only non-concordant result for -17p was a false positive that was detected by all 4 NGS CNV genotypers but not FISH, possibly suggesting a false negative FISH result. Conclusions: CANARy accurately identifies clinically significant CNVs in common hematologic neoplasms and could thus serve as a complementary approach to the current cytogenetic evaluation. CANARy uses data already generated from targeted sequencing panels and therefore does not impose additional work or cost. Low tumor fraction could be a limitation for CNV calling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor Derived Myelodysplastic Syndrome after Sex Matched Haploidentical Allograft for AML with Monosomy 7 in Both Diseases: Importance of Donor Chimerism Assessment By Two Independent Techniques

Donor-derived leukaemia is a rare outcome in patients following allogeneic hematopoietic cell transplant (HCT) accounting for ~ 0.1-5% of relapsed cases although the true incidence is likely underestimated. A 39-year-old male patient with a history of ulcerative colitis treated with mercaptopurine was subsequently diagnosed with therapy related acute myeloid leukaemia (AML) and karyotype showed 45,XY,-7. The patient achieved remission after two cycles of treatment and proceeded to a paternal haploidentical HCT. Complete remission with no detectable disease by flow cytometry and fluorescent in-situ hybridisation (FISH) was achieved by day 40 of transplantation with whole blood and T-cell chimerism of 97% as assessed by routine short tandem repeat (STR) chimerism analysis. With withdrawal of immunosuppression this increased to 100% in both fractions. At 11 months post-transplant evidence of low-level cytogenetic relapse emerged with monosomy 7 seen by FISH in 15/200 interphase cells. Chimerism remained stable at 99% with aspirate findings indicating 2-3% blasts and some trilineage dysplasia. Two subsequent marrows showed persistence of monosomy 7 by FISH with full donor STR chimerism. Although a possibility of donor derived myelodysplastic syndrome (DD-MDS) was considered, due to the monosomy 7, donor lymphocyte infusion (DLI) therapy was administered at 16 and 19 months post-transplant. Monosomy 7 persisted on FISH and standard cytogenetic analysis. To confirm validity of STR chimerism and to distinguish between DD-MDS and relapse of original disease, HLA typing by next generation sequencing (NGS) to exclude HLA loss related relapse and potentially guide rescue decisions was performed alongside NGS chimerism assessment. NGS chimerism utilises genetic markers with population independent discriminative power distributed across 17 chromosomes and further selected to allow sensitive detection combined with accurate and precise quantification of mixed chimerism. NGS chimerism is demonstrated to be sensitive enough to detect a minor DNA contributor comprising down to 0.5% of the sample, detection of low level mixed chimerism and therefore clinical utility in better predicting relapse compared to conventional techniques. Original stored donor cells were tested and showed no evidence of monosomy 7 by FISH. After confirming DD-MDS a second HCT was considered. Originally unsuccessful unrelated donor search was re-opened and wide family donor search initiated. With azacytidine therapy the patient achieved a short lived cytogenetic remission, but subsequently relapsed and progressed to AML with karyotype of 45,XY,-7,der(16)t(11;16)(q13;p13.1)[9]/46,XY[1] and died 33 months after HCT prior to initiation of a second HCT. This case was complicated by the presence of monosomy 7 detected by karyotyping in both the initial diagnostic t-AML and in the DD MDS. Monosomy 7 is the most common abnormality found in DD-MDS but a review of the literature available does not seem to indicate any cases where it was also present in the patient's initial diagnostic sample as a separate disease entity. The pathogenesis of DD-MDS remains unclear. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax (VEN) Plus Intensive Chemotherapy (IC) with FLAG-IDA in Patients (Pts) with Newly Diagnosed (ND) and Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Despite the high complete remission (CR) rates achieved with IC, relapse rates remain high underscoring the need for novel combination regimens capable of inducing deeper remissions via eradicating measurable residual disease (MRD). Recently, several studies have shown promising clinical activity of VEN when combined with different IC regimens in pts with ND AML with composite CR rates (CRc) above 90% and MRD-ve rates exceeding 80% (Wang 2022; Kadia 2021). FLAG-IDA + VEN has also shown significant clinical activity in pts with ND AML with a CRc rate of 89% and MRD-ve remission rate of 93% (DiNardo 2022). Methods: We conducted a single-center retrospective study to assess the clinical activity of FLAG-IDA + VEN in pts with AML. During induction, VEN was initially given for 14 days which was later on reduced to 7 days due to encouraging results by Kadia et al. Standard cytogenetic (CN) analysis was performed and molecular data was obtained using a 40-gene NGS platform. Responses were assessed using the modified International Working Group response criteria and included CR, CR with incomplete count recovery (CRi), morphologic leukemia-free state (MLFS), and partial remission (PR). Time-to-event analyses were estimated using the Kaplan-Meier method and compared using the log rank test. Results: From March 2020 to May 2022, 24 pts with AML (17 ND and 7 R/R) were treated using FLAG-IDA + VEN (Table 1). Median age was 48 years (range, 21-68) with male predominance (67%). Among the 17 pts with ND AML (8 de novo, 5 AML-MRC, 2 treated-secondary, 2 therapy-related), 3 had extramedullary disease (EMD). The most common ELN CN risk groups were complex (N=5), KMT2A-rearranged (N=4), other adverse (N=3), and diploid (N=3). Eight pts (47%) harbored a RAS mutation. Median time to initiation of therapy was 5 days (range, 2-14). Twelve pts (71%) [KMT2A-rearranged, N=4; complex CN, N=3; diploid, N=3; other adverse, N=1; other intermediate, N=1] achieved CR/CRi including 1 pt with EMD. Median time to ANC (≥ 500) and platelet (≥ 50,000) recovery were 29 days (range, 19- 42) and 25 days (range, 17- 42), respectively. Median number of consolidation cycles given were 1.5 (range, 1- 5). MRD status was available in 6 out of 12 pts and all 6 pts achieved MRD negativity using flow cytometry (<10-3). Median follow-up among ND AML pts was 4.3 months (mths) (range, 0.7- 13.2). Among the 12 responders: (1) 3 pts received allo-SCT [KMT2A-rearranged, N=2; (-7), N=1] after 7, 4, and 3 mths, respectively, and remain alive in CR 10+, 7.5+, and 3+ mths, respectively, after initiation of therapy, (2) 6 pts with ELN favorable (N=3), intermediate (N=2), and adverse (N=1) risk AML remain in CR/CRi for 6+, 4+, 1+, 3+, 2+, and 2+ mths, respectively, (3) 3 pts with ELN adverse risk AML (including 1 pt with de novo MLLr AML and EMD) achieved CR and relapsed after 0.7, 1.7, and 7.6 mths, respectively. One pt died due to progressive disease (PD) 4.7 mths after relapse and the remaining 2 pts are receiving salvage therapy and alive 4.3+ and 5.2+ mths after relapse, respectively. All nonresponders (N=5) had ELN adverse risk AML including 2 pts with EMD and 2 pts with treated secondary AML. All 5 nonresponders died due to PD including 3 pts who had no response to HMA+VEN based salvage therapy. Among the 7 pts with R/R AML, 6 were in salvage 1 and 1 pt received prior allo-SCT. Responses were observed in all pts: 4 CR, 1 CRi, 1 MLFS, and 1 PR. Median time to count recovery in R/R AML pts was 33 days (range, 24-78). Median follow-up in R/R AML pts was 6.2 mths (range, 1.5- 15.6). Of the 5 pts who achieved CR/CRi, 4 were bridged to allo-SCT and 1 pt died in CR after 3 cycles of consolidation due to sepsis. Of the 4 SCT recipients: (1) 2 pts relapsed 2 mths after allo-SCT and died 4 and 6.5 mths, respectively, after allo-SCT; (2) 2 pts (KMT2A-rearranged and NPM1mut) remain alive in CR for 8+ and 14+ mths, respectively. Two pts with complex CN achieved MLFS and PR after 1 cycle of therapy and died due to PD and fungal pneumonia after 5.3 and 1.5 mths of starting therapy, respectively. Median overall survival (OS) for pts with ND AML and R/R AML were 13.2 mths (95% CI: Not reached) and 6.2 mths (95% CI: 5-7), respectively (p=0.62; Figure 1). Among pts with ND AML, median OS was not reached among responders and was 2.6 mths in non-responders (p=0.002). Conclusion: FLAG-IDA + VEN is a feasible and active regimen in AML. Longer follow-up and larger multicenter studies are needed to confirm the efficacy of this regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Y Chromosome Material in Turner Syndrome.

BackgroundTurner Syndrome (TS) is a frequently identified chromosomal disease in humans characterized by short stature, sexual infantilism, streak gonads, primary amenorrhea, and a number of somatic anomalies. Approximately 55% of TS individuals have a nonmosaic 45,X karyotype. In addition, a cell line with a Y chromosome is present in 5% of patients, which is undetectable by the standard cytogenetic analysis. The identification of Y chromatin in some TS individuals has been associated with the development of gonadoblastoma. Therefore, it is important to exclude the presence of Y chromatin in TS individuals. In this study, it was planned to detect cases with mosaicism in terms of Y chromatin with the help of Y whole chromosome probes (WCP) from individuals with TS by fluorescence in situ hybridization (FISH) analysis.MethodologyForty-four patients with Turner syndrome, who were being followed up in the Genetics Unit, were contacted and invited for the study. Of the 44 invited patients, 28 responded to the invitation. In this study, it was planned to detect TS patients with mosaicism in terms of Y chromatin with the help of the Y whole chromosome probe.ResultsThe majority of the cases (71.42%) included in the study carried pure X monosomy, which is the classical Turner syndrome karyotype. Other structural X chromosome aberrations, in isolated or mosaic forms, were less frequently represented. Y chromosome sequences were searched in 28 cases with Turner syndrome by the FISH method using Y whole chromosome probe. Y chromosome sequence was detected in one (3.5%) case of 28 cases.ConclusionsIt is recommended that individuals with Turner syndrome be screened for Y chromatin. Detection of this will provide information and guidance to individuals with Turner syndrome, especially in terms of the risk of developing gonadoblastoma, with advanced clinical consultation. This study was conducted to emphasize the importance of this.

Balanced Chromosomal Rearrangements Associated with Hypoprolificacy in Australian Boars (Sus scrofa domesticus).

Balanced chromosomal rearrangements, mainly reciprocal translocations, are considered to be the causative agent of several clinical conditions in farmed pigs, resulting in hypoprolificacy and economic losses. Literature suggests that reciprocal translocations are heritable and can occur de novo. The prevalence rate of these balanced structural rearrangements of chromosomes differs from country to country and varies between 0.5% and 3.3%. The Australian pig population is descendent of a small founder population and has since been a closed genetic group since the 1980s. Hence, any incident of reciprocal translocation along with the pedigree of boars that contribute sperm for artificial insemination has the potential to have an economic consequence. To date, there has been no published account for screening of reciprocal translocation associated with hypoprolificacy in the Australian pig population. In this study, we performed standard and molecular cytogenetic analyses to identify evidence of chromosome rearrangements and their association with hypoprolificacy in a representative 94 boar samples from a commercial nucleus herd. We identified three novel rearrangements between chromosomes 5 and 14, between chromosomes 9 and 10, and between chromosomes 10 and 12. In addition, we also detected a reciprocal translocation between chromosomes 3 and 16 that has previously been detected in pig herds in France. The prevalence rate was 6.38% within the samples used in this study. All four rearrangements were found to have an association with hypoprolificacy. Further study and routine monitoring will be necessary to identify any further rearrangements that will allow breeders to prevent the propagation of reciprocal translocations from generation to generation within the Australian pig population.

Cytogenetic and Molecular Genetic Abnormalities of CIITA Gene in Patients with Primary Mediastinal (Thymic) Large B-Cell Lymphoma

Background. Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is an aggressive malignant lymphoproliférative disease which accounts for 2-3 % of all non-Hodgkin's lymphomas. In 40 % of PMBCL cases rearrangements of the MHC class II activator, i.e. CIITA gene, are observed. CIITA abnormalities lead to decreasing protein expression and surface expression of MHC class II, which results in lack of adaptive cell immunity targeted at tumor cells. Aim. To assess the rate and spectrum of cytogenetic and molecular genetic abnormalities of CIITA gene in PMBCL patients. Materials &amp; Methods. The study enrolled 37 patients with diagnosed PMBCL: 10 men and 27 women aged 21-61 years (median of 31 years). Sanger sequencing was performed in 36 patients. In 20 patients CIITA/16p13.13 FISH and in 15 patients standard cytogenetic analysis were carried out. Results. In 3 (8.3 %) out of 36 patients the sequencing method detected mutations impairing CIITA gene function, as well as microdeletion in exon 1, deletion and nucleotide substitution in a splice donor site. Multiple somatic variations in intron 1 were identified in 21 (58.3 %) patients: in 11 (52.4 %) cases there were deletions and single nucleotide variants (SNV); the other 10 (47.6 %) patients showed only SNVs. In 13 (61.9 %) out of 21 cases the abnormalities of promoter IV and/or alternative exon 1 were observed. In 5 (25 %) out of 20 patients the FISH assay identified CIITA gene translocation. Standard cytogenetic analysis detected complex karyotype in 7 (46.6 %) out of 15 patients. The comparison of data showed hypermutagenesis in 8 out of 10 patients with FISH-detected chromosome aberrations, and in 3 (37.5 %) of them complex karyotype aberrations were found as well. Conclusion. Molecular genetic methods identified different somatic variations in CIITA gene affecting its functionally important regions, which can be of special interest for further studying the biology of tumors, including PMBCL.

ALK-positive diffuse large B-cell lymphoma is associated with Clathrin-ALK rearrangements: report of 6 cases

AbstractExpression of ALK protein by lymphoid cells and the description of variant anaplastic lymphoma kinase (ALK) translocations have typically been restricted to cases of T-cell and null anaplastic large-cell lymphoma (ALCL). All such cases result from a novel fusion created by the ALK gene on chromosome 2p23 and NPM on 5q35 or other variant translocation partners. A rare variant of diffuse large B-cell lymphoma (DLBCL), originally described in 1997, was thought to overexpress full-length ALK in contrast to a chimeric protein characteristic of ALCL. However, full-length ALK protein lacks tyrosine kinase activity and thus the mechanism of oncogenesis has remained elusive. We describe 6 cases of ALK+ DLBCL characterized by a simple or complex t(2;17)(p23;q23) involving the clathrin gene (CLTC) at chromosome band 17q23 and the ALK gene at chromosome band 2p23. All cases were studied using fluorescence in situ hybridization (FISH), complemented in one case with standard cytogenetic analysis, multicolor karyotyping (M-FISH), and reverse transcriptase-polymerase chain reaction. These results clearly demonstrate that most cases of ALK+ DLBCL share the same mechanism of deregulated ALK expression. Moreover, these results demonstrate the presence of CLTC-ALK fusions in these tumors and extend the list of diseases associated with this genetic abnormality to include classical T-cell or null ALCL, ALK+ DLBCL, and inflammatory myofibroblastic tumors. (Blood. 2003;102:2568-2573)

Detection of Abnormal Pretransplant Clones in Progenitor Cells of Patients Who Developed Myelodysplasia After Autologous Transplantation

AbstractSecondary myelodysplastic syndromes (MDS) have been reported after autologous transplantation. It is not known whether the MDS results from the pretransplant conventional-dose chemotherapy or from the high-dose chemotherapy (HDC) used for the transplant procedure. We performed a multicenter, retrospective analysis of morphologically normal pretransplant marrow or stem cell specimens from 12 patients who subsequently developed myelodysplasia after HDC. To determine if the abnormal clone was present before HDC, we used fluorescence in situ hybridization (FISH) to detect the cytogenetic markers observed at the onset of posttransplant MDS. Cryopreserved, pretransplant bone marrow, peripheral blood stem cell specimens, obtained at the time of harvest, or archival smears were used. Standard cytogenetic analysis had been performed pretransplant in four patients, showing a normal karyotype. In 9 of 12 cases, the same cytogenetic abnormality observed at the time of MDS diagnosis was detected by FISH in the pre-HDC specimens. Our findings support the hypothesis that, in many cases of posttransplant MDS, the stem cell damage results from prior conventional-dose chemotherapy and may be unrelated to HDC or the transplantation process itself.

The Genotoxic Effect of Interventional Cardiac Radiologic Procedures on Human Chromosomes

In recent years, an important part of the ionizing radiation (IR) that human have been exposed for diagnostic purposes are interventional radiologic procedures. The X-rays and contrast media are used in angiography. The patients and staff members are exposed to X-ray during these procedures. While it is known that X-rays cause DNA damage and carcinogenesis, the effect of the contrast agent is still unknown. The aim of this study was to investigate the effect of X-rays and contrast agent on chromosomes of human patients. Peripheral blood samples were taken from 50 patients (30 males, 20 females, ages between 38-75 years). Chromosome analysis of peripheral lymphocytes in 50 patients were performed at 3 different periods; before the interventional radiologic procedure, 24 hours and 1-3 months after the procedure. Also, chromosome analysis was performed on 17 staff members working during interventional radiological procedures to investigate the effect of X-rays. Standard cytogenetic analysis techniques were used for this study. The frequency of chromosomal aberrations (CAs) was significantly higher in patients 24 hours after the interventional radiologic procedures than pretreatment (p=0,000). At the same time, CAs after 24 hours compared with those taken 1-3 months later, shown that the CAs were significantly reduced after 1-3 months (p=0,000). We also found that the frequency of CAs was also statistically higher in patients exposed to high radiation doses (p=0,042). Compared with the control group (n=30), CAs were found significantly higher in workers exposed to radiation. Our findings have shown that X-rays and contrast agents that used in interventional radiological cause chromosomal damage. For this reason, the dose of radiation to be given to the patient must be carefully selected. Due to the potentially high genetic damage of patients with coronary artery disease (CAD), the type and amount of medication to be given and the frequency of radiological diagnostic procedures to be performed should be meticulously adjusted.

Karyotype evaluation of repeated abortions in primary and secondary recurrent pregnancy loss.

To study the contribution of embryo chromosomal abnormalities in primary and secondary recurrent pregnancy loss (RPL) and to analyze the recurrence of chromosomal constitution in miscarriages from the same couple. Retrospective study of abortion karyotypes in RPL families based on the mother's primary or secondary RPL status (563 embryo specimens, 335 samples from primary, and 228 samples from secondary RPL). RPL was defined as two or more consecutive miscarriages. One hundred eight cases of recurrent embryo/fetal loss in 51 families were analyzed to assess the probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in both previous and subsequent pregnancy loss. The karyotypes of abortions were established using standard cytogenetic analysis, as well as interphase fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). The frequency of aberrations was 43.9% in abortions from primary RPL versus 52.6% in secondary RPL (p = 0.041). Women 35years of age or older were the main contributors to this difference. The odds ratio of a subsequent abortion having the same karyotype pattern (normal or abnormal) as the previous one was 6.98 (p = 0.0013). The frequency of abnormalities is higher in abortions from the secondary RPL versus primary RPL group, and this difference is due to the relative deficiency of miscarriages with abnormal karyotypes in older women with primary RPL. The probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in the previous and subsequent abortion is increased significantly compared with chance.

Использование трофобластов цервикального канала для неинвазивной пренатальной детекции частых анеуплоидий

The article presents an experience of isolation of trophoblast cells from cervical samples of pregnant women and demonstrates a possibility of determining aneuploid cells in cervical samples using FISH analysis. Objective. To study methods of effective isolation and reliable detection of trophoblasts for genetic pathology analysis. Patients and methods. The participants of the study were three pregnant women, who according to the findings of the combined first trimester screening test were referred to the group with a high risk for fetal aneuploidy. The terms of gestation varied from 12 to 14 wks. Immunocytochemical detection of trophoblast cells was performed with FITC-labelled HLA-G antibodies. FISH was performed with the use of locus-specific DNA probes on chromosomes 13/21 and a DNA probe on chromosome 18 centromere. The chromosome set of a fetus was verified by the results of standard cytogenetic analysis of semi-direct chromosome preparations from cytotrophoblast cells obtained by chorionic villi biopsy. Results. HLA-G-positive cells were found in all examined cytological specimens. FISH analysis with the use of DNA probes on chronomomes, whose trisomies are compatible with live birth, detected aneuploid cells in all three cases. Conclusion. The use of cervical trophoblasts for noninvasive collection of information about the genetic status of the developing fetus at present needs further study of effective and reliable methods of their isolation, detection and analysis. Key words: aneuploidy, throphoblast cells, NIPT, prenatal diagnosis, HLA-G

Svd-SRS: A Highly Specific Algorithm for Identifying Somatic Genomic Rearrangements in Haematological Malignancy

The detection of chromosomal rearrangements in haematological malignancies is important for providing appropriate clinical care. Many chromosomal rearrangements are detected by standard cytogenetic analysis (G-banding) but others are too subtle and evade detection by this method. Techniques such as PCR or FISH can be used to focus detection on specific rearrangements to confirm or refute their presence, but other less common but potentially significant rearrangements will be missed by gene-centric methods. Whole Genome Sequencing (WGS) has the potential to identify all possible genomic rearrangements and mutations in patients. Analysing WGS data is not trivial however, and currently available programs detect thousands of structural abnormalities in a single patient by comparison to the reference genome. Most of these abnormalities are simply not real somatic changes and have the potential to mislead and waste effort and resources in their confirmation. There is an urgent need for an analysis pipeline that is both sensitive and specific so that only the true somatic changes that cause the malignancy and influence its clinical course are detected. Here we describe SVD-SRS, a new algorithm for the detection of somatic genomic structural rearrangements in paired-end WGS data with high specificity, and without the need for a germline comparator. SVD-SRS was developed using a 64-core server to enable massively parallel data processing, but the algorithm is scalable and can be implemented on systems with fewer resources. SVD-SRS uses discordant and split reads to identify and model structural rearrangements in paired-end WGS data. SVD-SRS differs from other less specific algorithms because it performs a series of internal validation steps to identify only those discordant split reads that give reliable evidence of structural rearrangement. The orientation of reads with respect to their pairs is then used to categorise breakpoint sequences into the different types of structural rearrangement. To exclude structural polymorphisms, each putative breakpoint sequence is compared with publicly available databases of normal variants. To demonstrate the performance and utility of SVD-SRS, we analysed whole genome sequences of primary samples taken from patients with acute or chronic haematological malignancy, and compared these to that of MANTA, a commonly used structural variant caller. We also engineered 10 structural rearrangements into the reference genome at various levels to see if they would be detected. MANTA reported an average of 16,711 ± 800 structural variants in each primary sample. Using 16 cores, MANTA had a run time of 1h per sample. Filtering the output for only translocations with an allele frequency ≥30%, the output was reduced to &lt;100 events per sample but this removed clinically important validated rearrangements from the dataset. This demonstrates that filtering by read-depth is not adequate for improving the specificity of MANTA. It simply reduces the number of calls. In contrast, SVD-SRS reported &lt;10 events for each sample, with a run time of &lt;12h using 16 cores. The run time per sample is longer compared with MANTA because of the validation steps taken to select appropriate reads and exclude structural polymorphisms. Notably the massively reduced number of reported variants makes scrutiny of the data much more feasible in a clinical setting. SVD-SRS detected all rearrangements present in the initial cytogenetic report of the samples tested. This included t(9;22)(q34;q11) BCR/ ABL1, inv(3)(q21;q26) GATA2/ MECOM, and t(8;14)(q24;q32) IGH/ MYC in CML, ALL, and Burkitt's Lymphoma samples, as well as the immunoglobulin heavy chain recombination events important for ALL and CLL prognosis. SVD-SRS also detected 1 or 2 additional rearrangements of clinical importance (e.g. del(9p21) CDKN2A/ CDKN2B) in 30% of the samples analysed. All additional rearrangements were validated by PCR. For the engineered sample, SVD-SRS detected all but one event which was present at a very low level, whilst MANTA identified all events as well as an additional 20 that were not introduced into the reference genome in the first place. Based on these data, SVD-SRS has massively improved the specificity of structural variant detection compared with MANTA, and is suitably sensitive and specific to replace cytogenetic techniques in clinical practice. Disclosures No relevant conflicts of interest to declare.