The Significance of Chromosome 11 Abnormalities in Patients with AML

Introduction: At the time of diagnosis AML the finding of KMT2A gene rearrangement qualifies patient to the group with adverse cytogenetic risk according to ELN 2017. In many hematooncological diseases including AML abnormalities concerning another regions of chromosome 11 are detected. However, their significance for outcome, CR rate, OS is still unknown. The main aim of this study was analysis of chromosome 11 abnormalities in AML patients, focusing on these without KMT2A gene rearrangement and its influence on patients outcome. Methods: Karyotype analysis from bone marrow aspirate was performed for patients with chromosome 11 abnormalities (abn11, n=36) and compared to two groups: with complex (CK, n=20) and normal karyotype (NK, n=44). The basic clinical and laboratory data including cytogenetic (karyotype and FISH method) and presence of TP53 deletion were analyzed to characterize in detail the group of patients with chromosome 11 abnormalities. Patients were treated with intensive chemotherapy, in 33% of them allogeneic cell transplantation (alloHCT) was performed. Results: In patients with abn11 the most frequent type of aberration was translocation, 11q23(+)/KMT2A(-) and 11q10-25 as a main region involved in aberrations, with minimal narrowing of region to 11q22. In patients with abn11 and translocation (excluding t(9;11)) there was higher frequency occurrence of 1 to 2 additional abnormalities (p=0.033) and lower frequency of complex karyotype existence (p=0.026). The gain of genetic material from chromosome 11 was observed in 33% of patients with abn11 group and the minimal amplified region was 11q22. Furthermore, in patients with amplification of chromosome 11 region, different additional cytogenetic abnormalities (such as monosomy 5 (p=0.045) and 18 (p<0.001)) were found in comparison to patients with other abnormalities of chromosome 11. Additional KMT2Agene copies were often observed in patients with 11q10-25 (p=0.013) and 11pter-qter (p=0.022) region involvement. The lowest frequency of TP53 deletion was in NK group (p=0.002) but comparable in patients with CK and abn11 without impact on OS and CR. The FISH analysis did not revealed KMT2A gene rearrangement in group with normal karyotype. There was particular attention to 11q23 region in patients with AML which enabled abnormality detection in GTG analysis, furthermore KMT2A rearrangement was confirmed or excluded by FISH method. In few cases FISH analysis allowed verification of KMT2A gene status in case of atypical hybrydization pattern occurrence. Leukocyte counts (p<0.001) and type of AML (de novo, secondary, therapy-related, p=0.022) differs in patients with abn11 in comparison to remained studied subgroups (Table 1.). The lowest blast percentage was observed in patients with 11p rearrangement (p=0.045). There were no significant differences in OS between all 3 groups of patients. CR was achieved in 15% in a CK group and it was significantly lower than in NK and abn11 group (p<0.001). Furthermore alloHCT improve OS in all treated patients (p<0.001). Additional genetic material from chromosome 11 occurrence had negative impact on OS (p<0.001) and CR (p=0.007). Patients with abn11 who achieved CR had lower median age (p=0.01) compared to these without CR. The lowest percentage of CR in abn11 group was observed in patients with additional KMT2A gene copies (p=0.008), 11q10-25 region involvement (p=0.036), HSR (homogeneously staining region) and aneuploidy (p=0.04). Conclusions: In summary, the most frequent region involved in chromosome 11 aberration was 11q22. Additional material derived from chromosome 11 had negative impact on CR and OS. Cytogenetic and molecular analysis in patients with AML are needed to detect most of abn11. In the era of targeted therapies their identification is very important to qualify the patient for more effective treatment. Both cytogenetic and molecular techniques widespread knowledge about cancer biology, however, classic cytogenetic is still "gold standard" and crucial method in AML diagnosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Three-Color FISH Method: Dose-Effect Curves for Translocations in Peripheral Blood Lymphocyte Cultures after Gamma-Irradiation In Vitro

Purpose: Plotting dose-effect curves for translocations identified using the tricolor FISH method based on the results of cytogenetic analysis of cultures of peripheral blood lymphocytes of healthy donors after in vitro gamma irradiation. Material and methods: Venous blood was obtained from three donors (2 men and 1 woman aged from 28 to 41 years) and subjected to in vitro gamma irradiation from a 60Co source at doses of 0.10; 0.15; 0.25; 0.35; 0.50; 0.75; 1.00; 1.50; 2.00 and 3.00 Gy at 37 ° C (dose rate 0.5 Gy / min). For tricolor FISH staining, two different sets of DNA probes were used for chromosome pairs 1, 4, 12 and 2, 3, 8. Metaphases with a quasi-diploid number of chromosomes (40-46) and a complete set of all FISH-stained chromosomes, taking into account their total length, were selected for analysis. Differentiation of stable and unstable cells was also carried out. In the cytogenetic analysis, traditional terminology was used with the designation of translocations as reciprocal (complete, two-sided), non-reciprocal (terminal, incomplete, or unilateral), or interstitial. Results: The obtained numerical data were used to statistically compare the frequencies of FISH-recorded translocations when using different sets of DNA probes, when calculating of chromosome aberrations were in all (unstable and stable) and stable metaphase cells, when comparing of the frequencies of FISH-recorded translocations and dicentrics, and assessing of the contribution of the level of translocations between FISH-stained chromosome pairs in the total translocation frequency. The plotted dose-effect curves generally corresponded to the linear-quadratic form. Conclusion: Dose dependences obtained for translocations using two different selected tricolor sets of DNA probes did not differ statistically significantly. At the same time, cytogenetic analysis of only stable metaphase cells revealed a tendency to register lower levels of translocations than when analyzing all cells (unstable and stable ), at the highest doses of 2 and 3 Gy. The levels of dicentrics formed with the participation of FISH-stained chromosomes were significantly lower than the number of observed translocations. The quantitative contribution of translocations between FISH-stained pairs of chromosomes turned out to be very low, which clearly does not contribute to an increase in the sensitivity of the FISH method of retrospective dose estimation as compared to its one-color version. At the same time, the three-color FISH-staining makes it possible to identify such variants of chromosomal rearrangements that are not recorded using the one-color FISH method.

Intrachromosomal Amplification of Chromosome 21 in Childhood Acute Lymphoblastic Leukemia: Study of 3 Cases

Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. The presence or absence of a characteristic genetic abnormality usually observed in childhood ALL plays a very important role in determining the prognosis and stratification for treatment. Intrachromosomal amplification of chromosome 21 (iAMP21) is an uncommon high-risk chromosomal abnormality than can occur only in 2% of childhood B-cell precursor lymphoblastic leukemia. Molecular genetic analysis and the fluorescence in situ hybridization (FISH) technique are the basic methods used to detect the presence of the most common genetic abnormalities, the presence or absence of which has an impact on the patient’s classification into the appropriate risk group. This work presents 3 BCP-ALL iAMP21-positive patients who were detected during routine genetic diagnostics using the FISH method and microarray test. iAMP21 is associated with a poor prognosis and high risk for relapse. Children with B-cell precursor lymphoblastic leukemia with this genetic entity are associated with a delayed treatment response. The FISH method and single-nucleotide polymorphism array provides a useful method to detect characteristic genetic changes.

Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

ANALISIS KEBERLANJUTAN PENGELOLAAN LIMBAH DI INSTALASI KARANTINA HEWAN (IKH) RUMINANSIA BESAR

ABSTRACT Indonesia regulates post-entry observations of slaughter animals through the Animal Quarantine Installation (AQI). For the continuation of the existence of AQI, it is necessary to carry out an analysis of the sustainability of AQI waste management, because errors in waste management can cause disease and environmental pollution. The purpose of this study is to evaluate and determine the sustainability of AQI based on 5 dimensions, namely the dimensions of ecology, economics, technology, social, and institutions. AQIs taken as research objects are one government AQI and one private AQI. The study was conducted by observation. The results of the observations were analyzed using a modification of the Rap-fish method with Multidimensional Scaling called Rap-AQI. The results showed the sustainability of private AQI in multidimensional aspects showing a sustainability index of 57.47, each indicated from the dimensions of ecology (54.17), economy (70.12), social (57.47), technology (54.89), and institutional (50,73). Whereas the government's AQI showed unsustainable results with a sustainability index of 45.02, each from ecological dimensions (49.24), economic (45.30), social (55.77), technology (29.27) and institutional dimensions (43.53). Leverage attribute analysis shows that of 54 existing attributes, there are 12 sensitive attributes as a key factor in the sustainability of AQI waste management. Keywords: Sustainability, animal quarantine installation, atribute, dimention ABSTRAK Indonesia mengatur pengamatan pasca-masuk hewan potong melalui Instalasi Karantina Hewan (IKH). Pemerintah telah melakukan kebijakan untuk pencegahan atau meminimalkan risiko penyebaran organisme penyakit hewan dan zoonosis pada kegiatan impor hewan dipintu masuk yaitu IKH. Untuk keberlanjutan keberadaan IKH, perlu dilakukan analisis keberlanjutan pengelolaan limbah IKH, karena kesalahan dalam pengelolaan limbah dapat menimbulkan dampak penyakit dan pencemaran lingkungan. Tujuan dari penelitian ini adalah melakukan evaluasi dan menentukan keberlanjutan IKH berdasarkan 5 dimensi, yaitu dimensi-dimensi ekologi, ekonomi, teknologi, sosial dan kelembagaan. IKH yang diambil sebagai objek penelitian adalah satu IKH pemerintah dan satu IKH swasta. Penelitian dilakukan dengan pengamatan. Hasil pengamatan dianalisa dengan menggunakan modifikasi metode Rap-fish dengan Multidimensional Scaling yang disebut Rap-IKH. Hasil penelitian menunjukkan keberlanjutan IKH swasta dalam multidimensi aspek menunjukkan indeks keberlanjutan 57,47, masing-masing ditunjukkan dari dimensi ekologi (54,17), ekonomi (70,12), sosial (57,47), teknologi (54,89), dan kelembagaan (50,73). Sedangkan IKH pemerintah menunjukkan hasil yang kurang berkelanjutan dengan indeks keberlanjutan 45,02, masing-masing dari dimensi ekologi (49,24), ekonomi (45,30), sosial (55,77), teknologi (29,27) dan dimensi kelembagaan (43,53). Analisis leverage atribut menunjukkan bahwa dari 54 atribut yang ada, terdapat 12 atribut sensitif sebagai faktor kunci keberlanjutan pengelolaan limbah IKH. Kata kunci: Keberlanjutan, instalasi karantina hewan, atribut, dimensi

Development of a fixation-free fluorescence in situ hybridization for the detection of Salmonella species

Salmonella is one of the most important infectious bacteria causing severe gastroenteritis and deaths in humans and animals, and the prompt diagnosis is crucial for effective control and treatment. The detection of Salmonella still depends principally on culture-based methods, which is time-consuming and laborious. Recently, polyhexamethylene biguanide (PHMB) was discovered to have cellular delivery properties and its combination with the fluorescence in situ hybridization (FISH) method was exploited for oligomer delivery and the rapid detection of Salmonella spps in this study. Cell-associated fluorescence was quantified using Volocity® 3-D image analysis software (Volocity 6.3, PerkinElmer, Inc.). PHMB complexed with fluorophore—labelled species-specific oligomers permeabilized freshly grown viable strains of Salmonella cells and mediated strong cell-associated fluorescence signals. This strategy further enabled a fixation-free protocol and hybridization in a single reaction. Using the modified FISH method, monoculture Salmonella strains were validated as well as detected in artificially contaminated water and milk within a turnaround period of 5 h. The method was observed to be comparable with the standard FISH technique (sFISH; P > 0.05). The findings suggest that fixation-free delivery and hybridization of oligomers using PHMB can provide a simplified and prompt strategy for Salmonella detection at the species level, and promote early management responses to the disease and appropriate antimicrobial therapy.