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*Corresponding Author: Pina Trivedi, Senior Scientific office & Head, Cytogenetic Laboratory Department of Cancer Biology the Gujarat Cancer & Research Institute, Asarwa, Ahmedabad-380016, INDIA.
Citation: P Trivedi, D Patel, P Varma, M Kazi. (2022). Identification of a Novel Unbalanced t(4;8) (q12; q24) in Paediatric Acute Myeloid Leukemia M2 with AML-ETO Negative Patient by Fluorescence in Situ Hybridization Technique. Journal of Clinical and Laboratory Research. 5(3); DOI:10.31579/2768-0487/074
Copyright: © 2022 Pina Trivedi. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 28 January 2022 | Accepted: 20 February 2022 | Published: 02 March 2022
Keywords: myeloid leukemia; situ hybridization technique; leukemogenesis
Identification of chromosomal abnormalities in patients with Acute Myeloid Leukemia (AML) has contributed substantially to our current understanding of the molecular pathogenesis underlying leukemogenesis, and risk-stratification. Based on molecular abnormalities both influences treatment strategies and aids in determining prognosis. While over 300 established mutations have been documented in AML, the enhanced availability of genetic analysis and the increase in awareness of uncommon chromosomal translocations have made it possible for rare, apparently unique translocations to become recognized and to ultimately gain prognostic significance. Hence, we present a case of AML with a novel, t(4;8) involving breakpoints previously undescribed. Although the patient required second induction, first remission was ultimately achieved. While the prognostic significance of this translocation is not fully elucidated, it is our hope that documentation of this patient’s presentation will help to characterize the significance of a yet undefined cytogenetic abnormality in AML.
The most common form of the acute leukemias is an Acute Myeloid Leukemia (AML), with nature of complexity, heterogeneity and with marked genetic, epigenetic, and phenotypic variation [George M. Jehaa, Tiffany Wesley, Vince D. Cataldo 2020]. In most cases of AML, Chromosomes of malignant cells harbour specific, non-random, and often recurrent abnormalities which have important implications in terms of clinical and pathological presentation, prognosis, and therapeutic response. AML in children is a rare and heterogeneous disease, with an incidence of 7 cases per million children younger than 15 years. Survival rates increased upt to 70% due to intensive therapy in combination with effective supportive therapy in high-income countries, [Amanda de Lourdes Nunes et al,Ursula Creutzig et al 2012].
According to cytogenetic abnormalities chromosomes are grouped in three prognostic categories: favorable, intermediate, and adverse. Some of these abnormalities are common and others are rare. Rare cytogenetic abnormalities include aberrations of chromosomes 3, del (5q),- 5 and − 7 .Cytogenetic and molecular data are recognized as the most valuable prognostic factors in AML both in National Comprehensive Cancer Network (NCCN) and European Leukemia Net (ELN) risk stratification models [ANUDISHI TYAGI et al 2018 & Matahi Moarii et al 2017].
In this case, we describe a report of AML-M2 patient with cytogenetic and FISH study using Bone marrow sample. Cytogenetic results revealed that there was a translocation between chromosome 4 and 8 in all 20 metaphases analyzed. Patient's karyotype analysis revealed 46, XY, t(4;8)(q12;q24)[20]. Karyotype results and FISH results were compared using Whole Chromosome Paint (WCP) FISH probes and Locus Specific Identifier (LSI) RUNX1/RUNX1T1fusion Dual color dual fusion (DCDF) probe.
We report a patient with AML-M2 who developed t(4;8)(q12;q24) prior to the course of therapy. A 2-year-old male child suffering from low grade fever, weakness, join swelling of bilateral dorsum of hand to Gujarat Cancer and Research Institute, Ahmadabad, India.
On admission the laboratory investigations revealed White Blood Cell count 67.63 X103/cmm, Red Blood Cell count 4.34X106/cmm, Haemoglobin-8.8 gm/dl, and Platelets 231 X103/cmm, Myelocyte 14 %, Polymorphs 56%, and Lymphocytes 8%, Eosinophils 3%. Bone marrow report showed hypercellular with altered M: E ratio. PAS Stain Negative, Sudan Black Positive, Blast Cells 45 %, Promyelocytes 5 %, Myelocytes-M 10 %, Metamyelocytes- 2 %, Band Cells 2 %, Polymorphs- 4 %, Eosinophils- 3 %, Lymphocytes- 4 %, Inter Normoblasts5 %, Late Normoblasts 8 %, Reticulum Cells- 2 %. It shows proliferation of blast cells (45%). These blasts are medium to large in size, having high N:C ratio, fine chromatin & moderate amount of cytoplasm, having fine granules in few. Myeloid & erythroid precursors are suppressed. Megakaryocytes are present. RBCs are Hypochromic microcytic. Marked leukocytosis with blast cells. Blast-11, Promyelocyte-05, Myelocyte-14, P-56, L-08, M-03, E-03. Platelets - Adequate. Diagnosis: Acute Myeloid Leukemia (AML-M2/M4). X-Ray Reports of chest was normal. Ultra-sonography reports of Abdomen pelvis were normal. FLT3/ITD mutation by RFLP method was negative from peripheral blood sample.
The patient was treated with Cytarabine 100 mg, Etoposide 100 mg, Filgrastim 300 mg, Mercaptopurine 50mg, Vincristine 1 mg, Doxorubicin 10mg and achieved remission.
Present study was approved by the institutional review board and patients’ general consent was taken.
Conventional Cytogenetics
Bone marrow sample was collected ascetically in Sodium Heparinized vaccuate. For conventional cytogenetic study, short term culture was carried out as per standard protocol and slides were banded using Giemsa Trypsin G banding technique. Good morphology metaphases were captured in Zeiss automatic karyotyping system and analysis using IKAROS software and karyotype description was done using ISCN 2016 guidelines.
Fluorescence in situ hybridization (FISH):
Using Epi-fluorescence microscope (AXIO Imager.Z2, Zeiss, USA) equipped with appropriate filter sets, image capturing, and processing were carried out using an ISIS FISH imaging system (MetaSystems, Germany). FISH was performed using RUNX1/RUNX1T1 LSI probe. In LSI RUNX1/RUNX1T1 probe, RUNX1 gene was tagged with Spectrum green and RUNX1T1 gene tagged with Spectrum Orange.
Conventional Cytogenetic:
Conventional chromosome analyses at diagnosis of GTG banded metaphase were carried out. Total 20 metaphases were karyotyped. All metaphases showed 46,XY,t(4;8)(q12;q24)[20] (Figure.1 A).
FISH:
FISH results with RUNX1/RUNX1T1 probes results revealed metaphases with 2O2G signals which was observed which showed that there was no fusion for RUNX1/RUNX1T1 gene (Figure.2). FISH result was nuc ish(RUNX1/RUNX1T1) X2[200].
As this is an unknown translocation in AML, a gene specific probe for this specific translocation was not utilized. So, the WCP FISH was carried out to determine the nature of the translocation. The WCP FISH probes for chromosome 4 Spectrum Green (SG) and chromosomes 8 with Spectrum Orange (SO) were applied on metaphase cells as per manufactures instructions. Results of WCP FISH revealed that there was unbalanced translocation between chromosome 4 and 8. Part of q arm of chromosome 4 was observed attached to the q arm of chromosome 8 and confirmed that t(4;8)(Figure 1B).
Conventional cytogenetics can detect structural and numerical cytogenetic abnormalities in 70%-80% of children with AML. Certain fusion genes, products from cryptic translocations, or loss of chromosome material can only be reliably detected using FISH. The diagnostic cytogenetic workup in children and adults is similar. The most frequent chromosomal abnormalities in children with AML include t(8;21)(q22;q22), inv(16)(p13.1q22) (together referred as core binding factor [CBF]-AML, t(15;17)(q22;q21)/PML-RARA, and 11q23/MLL-rearranged abnormalities (up to 25%), which together account for approx. 50% of pediatric AML, a much higher frequency than in adults. Additional abnormalities that are more predominant in pediatric AML are, for example, t(1;22)(p13;q13)/RBM15(OTT)-MKL1(MAL). Further types are the cryptic abnormalities t(7;12)(q36;p13)/ETV6(TEL)- HLXB9(MNX1), which are strongly associated with +19 and t(5;11)(q35;p15.5)/NUP98-NDS1, predominantly found in cytogenetically normal AML (CN-AML). However, both translocations have not yet been included in the WHO 2008 classification [Creutzig et al 2012].
In present study we come across novel translocation (4;8)(q12;q22). The gene present on 4q12 is PDGFRA & several chromosomal rearrangements generating fusion genes causing PDGFRA activation have been described in a variety of uncommon hematologic disorders that are often accompanied with a related condition called hypereosinophilic syndrome. These rearrangements activate PDGFRA by fusion to various partner genes: STRN (2p24) in the , FIP1L1 (interstitial 4q12 deletion), CDK5RAP2 (9q33) in the , KIF5B (10p11) in the , ETV6 (12p13) in the , and BCR (22q11). [Baxter et al., 2002; Gotlib et al., 2008]. PDGFA has transmembrane receptor protein tyrosine kinase activity and acts as a cell-surface receptor for members of the platelet-derived growth factor family: PDGFA, PDGFB and PDGFC, which are mitogens for fibroblasts and cells of mesenchymal origin. It plays an essential role in the regulation of many biological processes including cell proliferation, survival, differentiation, and cell migration & plays a role in platelet activation, wound healing, and angiogenesis [Demoulin et al., 2012; Heldin et al., 20103]. MYC gene located on 8q24 and present predominantly in the nucleus & expressed in almost all proliferating cells in embryonic and adult tissues; expression correlates with cell proliferation; abnormally high expression is found in a wide variety of human cancers. Its main role in Burkitt lymphoma, diffuse large cell Lymphoma, multiple myeloma, lung cancer, breast cancer colon cancer and prostate cancer & it’s expressed by different mechanisms like chromosomal translocation, amplifications, point mutations, epigenetic reprogramming, enhanced translation and increase protein stability. In 70% of cancers its expression is deregulated or elevated [Adrian Krygier et al 2020].
Outcome is of paediatric patients mainly depends on the initial response to treatment and chromosomal aberrations. Combinatorial therapy of Anthracycline- and Cytarabine-containing chemotherapy and stem cell transplantation in selected genetic high-risk cases or slow responders. Relapse observed in, ~30% of all pediatric AML patients, whereas death is observed in 5%–10% of the patients due to disease complications or the side-effects of the treatment [Jasmijn D. E. de Rooij et al 2015]. Our patient received standard induction therapy and Etoposide and 6 thioguanine and achieved complete remission. Standard induction therapy comprises 3 days of an Anthracycline (e.g. Daunorubicin at least 60 mg/m2, idarubicin 10-12 mg/m2, or the Anthracenedione Mitoxantrone 10-12 mg/m2) and 7-10 days of Cytarabine (100-200 mg/m2 continuously or twice daily intravenously, ie, “3_7” or “3_10”). With these regimens, ~85% of children and adolescents achieve complete remission (CR). Although a third drug, such as Etoposide or 6-thioguanine, is commonly included in induction, their benefit has not been proven.
Age is an important factor among children, as indicated by the age-specific FAB/WHO subtypes. Infant AML was historically defined as AML occurring in children under 1 year of age, but it has now been extended to include all children under 2 years, because they share the same clinical and biological profiles [Julie Quesada et al 2021]. For example, they include a higher proportion of acute megakaryoblastic leukemia (AMKL, M7), whereas certain cytogenetic abnormalities are exclusively identified in this age group, such as t(1;22)(p13;q13)/RBM15- MKL[Creutzig, U. et al 2012; De Rooij et al 2015; Harrison et al 2010; Von Neuhoff et al 2010].
Till date in Mitelman database, 71,734cases and 32,721gene fusion for the chromosomal aberration have been reported [July 15, 2021]. Moreover, t(4;8)(q12;q24) with different regions have been observed in 11 cases in different malignancies but not observed in AML-M2. So, our case is the novel finding. Although this patient required second induction, first remission was ultimately achieved. While the prognostic significance of this translocation is not fully elucidated, it is our hope that documentation of this patient’s presentation will help to characterize the significance of a yet undefined cytogenetic abnormality in AML.
To identify the exact breakpoints, present in AML and MDS and to identify fusion genes because of the unbalanced translocation array-CGH analysis required.
Targeted therapy may enhance anti-leukemic efficacy and minimize treatment-related morbidity and mortality but requires detailed knowledge of the genetic abnormalities and aberrant pathways involved in leukemogenesis. So, the presence of certain genetic abnormalities, usually detected by Fluorescence in Situ Hybridization (FISH) analysis, Polymerase Chain Reaction (PCR) and next generation sequencing makes it possible to risk stratify patients at the time & these efforts towards future personalized therapy in pediatric AML.
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