1. Group5.pdf

BMS4470A Histopathology Spring 2023-2024 Acute Promyelocytic Leukemia Jaber Ahmed Alnoamani 1082333 Submitted to: Dr Merin Thomas Sulthan Mujeeb Rahman 1078838 Date of Presentation: 29th May 2024  Introduct...

BMS4470A Histopathology Spring 2023-2024 Acute Promyelocytic Leukemia Jaber Ahmed Alnoamani 1082333 Submitted to: Dr Merin Thomas Sulthan Mujeeb Rahman 1078838 Date of Presentation: 29th May 2024  Introduction Diagnosis 01 04 Pathophysiology Treatment 02 05 Clinical Presentation Conclusions 03 06 01 Introduction  Acute Promyelocytic Leukemia (APL) Unique subtype of acute myeloid leukemia 10-15% of all AML cases (AML) characterized by accumulation of M0 -> M7 promyelocytes French American British (FAB) System M3 Chromosomal Translocation t(15;17) PML/RARα Blood Cell Lineage An In Vitro Model of Hematotoxicity: Differentiation of Bone Marrow–Derived Stem/Progenitor Cells into Hematopoietic Lineages and Evaluation of Lineage‐Specific Hematotoxicity - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Hierarchy-of-hematopoietic-cells-during-normal-differentiation-of-bone-marrow-derived_fig1_324848224 [accessed 27 May, 2024] 2. Pathophysiology Reciprocal translocation involving chromosome 15 and 17 t(15;17) PML RARα Retinoic acid-alpha Promyelocytic gene on chromosome leukemia gene on 17 chromosome 15 PML/ RARα Head-to-tail fusion encodes abberrant retinoid receptor t(15;17) translocation in APL Acute Promyelocytic Leukemia (APL): A Review of the Classic and Emerging Target Therapies towards Molecular Heterogeneity - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Chromosomal-translocation-between-chromosomes-15-and-17-Acute-promyelocytic-leukemia-is_fig1_368020769 [accessed 26 May, 2024] 3. Clinical Presentation Bruising and Blood clots Bleeding Hematuria Fatigue, Anemia Disseminated intravascular coagulation (DIC) Petechiae Thromboembolism Epistaxis Pupura Intracranial hemorrhage Dyspnea  04 Diagnosis  Peripheral blood smear A blood smear is used to evaluate RBC, WBC and platelet noting any abnormal differences in size, shape, or other physical appearances. The blood smear and bone smear are usually a confirmatory tests after CBC for diagnosing APL. Peripheral Blood smear procedure Sample Slide Staining Microscopy Collection Preparation Wright-Giemsa, Myeloperoxidase (MPO), are the main stains used in the diagnosis and assessment of APL  Procedure Retrieved from https://www.vet.cornell.edu/animal-health-diagnostic- center/laboratories/clinical-pathology/samples-and-submissions/hematology  Automated Smear Process 1. Smear Preparation: The automated system takes a small amount of blood and spreads it evenly across a glass slide to create a thin smear 2. Staining: The slides are then automatically stained using standardized protocols. The most common stains used are Wright-Giemsa or similar 3. Digital Imaging :The stained slides are scanned by the automated system, capturing high-resolution digital images of the blood smear. Automated smears represent a significant advancement in hematology, combining speed, consistency, and advanced image analysis to enhance the diagnostic process for conditions like APL and other blood disorders.  Normal blood smear morphology 1. (RBCs): Biconcave discs, generally round with minimal variation (anisocytosis) and no significant shape abnormalities (poikilocytosis). 2. Neutrophils: Segmented into 2-5 lobes 3. Lymphocytes: Nucleus is round and occupies most of the cell 4. Monocytes: Large, often kidney-shaped or folded, with less dense chromatin 5. Eosinophils: Typically, bilobed ,reddish-orange granules 6. Basophils: Lobed but often obscured by granules. 7. Thrombocytes: Small, anucleate cytoplasmic fragments. Retrieved from https://www.medical-labs.net/normal-blood-smear-649/ Normal Blood Smear Retrieved from https://imagebank.hematology.org/imageset/668/normal- peripheral-blood-smear Peripheral blood smear workup of acute promyelocytic leukemia (APL) A high number of abnormal promyelocytes, characterized by their large size, abundant cytoplasmic granules, and sometimes the presence of Auer rods. These promyelocytes exhibit a high nucleus-to-cytoplasm ratio with irregularly shaped nuclei. Pancytopenia is often associated with sporadic aberrant promyelocytes in circulation. Thrombocytopenia Peripheral blood smear workup of acute promyelocytic leukemia (APL) Peripheral blood film showing circulating blasts, bilobed , occasionally hypergranulated cells and auer rods (Wright stain x 1000) Retrieved from https://www.grepmed.com/images/3997/apml-promyelocytic-smear-rods-clinical  Bone Marrow Smear Diagnostic procedure called a bone marrow smear, a small amount of bone marrow is often aspirated from the hip bone and spread out onto a glass slide for microscopic analysis. The cellular makeup of the bone marrow, including the many phases of blood cell development (hematopoiesis), is clearly seen in this smear. It makes it possible to evaluate the shape of the cells, identify abnormal cells, and examine the cellularity and architecture of the marrow.  Normal bone smear morphology The bone marrow is composed of a mix of hematopoietic cells and fat cells. Some cell that can be observed under microscope include: Megakaryocytes Prolymphocytes: Intermediate stages with a slightly more condensed chromatin. Band Cells: Immature neutrophils with a horseshoe-shaped nucleus. Proerythroblasts: Large cells with round nuclei and prominent nucleoli. Retrieved from https://medicine.nus.edu.sg/pathweb/normal-histology/bone-marrow/  Bone marrow smear workup of APL Key Findings in APL Hypercellular, and APL promyelocytes account for about 30% of the myeloid cells in the classic variant. Promyelocyte has a creased, folded, bilobed, kidney- shaped, or dumb-bell shaped nuclei with a high nucleus-cytoplasmic ratio, fine chromatin, and prominent nucleoli. Decrease in the number of mature granulocytes and erythroid precursors. Increase condensed chromatin. Increase in the number of pseudo-Pelger-Huet cells Many violet granules which coalesce to form Auer rods Reference for findings: Syed Zaidi, M.D.. APL with PML-RARA. APL with PML-RARA. Last author update: 1 February 2013 Bone marrow smear of APL Amoth H, Perry AM. APL with PML::RARA. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/leukemiaAPL.html. Accessed May 27th, 2024. Karyotyping: Karyotyping provides essential genetic information that can significantly impact the diagnosis, prognosis, and treatment strategies for cancer patients. Karyotype tests are types of genetic tests. It examines the size, shape, and number of chromosomes in a sample. In all cells except, for egg and sperm cells there is a set of 46 chromosomes arranged in 23 pairs.  Steps in Karyotyping Cell Collection Cell Arrest Fixation Staining 1 2 3 4 5 6 7 8 Cell Culture Hypotonic Slide Microscopy Treatment Preparation Karyotyping Steps A normal karyotype indicates the typical chromosomal setup without any numerical or structural abnormalities. Cytogenetic Techniques in Diagnosing Genetic Disorders - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/A- karyotype-of-a-normal-male-46-XY-Reproduced-courtesy-of-Human-Genome-Centre_fig1_221920029 [accessed 27 May, 2024]  Karyotyping workup of acute promyelocytic leukemia (APL) Karyotyping is essential in the initial workup of acute promyelocytic leukemia (APL) to detect rare molecular subtypes and additional cytogenetic abnormalities, such as the characteristic t(15;17) translocation. This translocation involves the PML gene on chromosome 15 and the RARA gene on chromosome 17, resulting in a fusion gene that produces the PML- RARα protein. Fluorescence in situ hybridization (FISH) FISH is a powerful molecular cytogenetic technique that uses fluorescent probes to detect and localize specific DNA sequences on chromosomes. Sample Probe selection Bone marrow or blood Dual probes to bind to smear breakpoint Hybridization Visuilization Probes bind to the DNA The hybridized sample is sequence examined under a fluorescence microscope a. Two red and 2 green signals indicating lack of rearrangement of chromosomes 15 and 17. b. the dual color and fusion of the colors indicates PML/RARA fusion gene Wrede, J., Sundram, U., Kohler, S. et al. Fluorescence in situ hybridization investigation of cutaneous lesions in acute promyelocytic leukemia. Mod Pathol 18, 1569–1576 (2005). https://doi.org/10.1038/modpathol.3800465 FISH Fluoresence in situ hybridization pattern observed on metaphase cells and nuclei. PML, promyelocytic leukemia; RARA, retinoic acid receptor α Venci, A., Mazza, R., Spinelli, O., Di Schiena, L., & Bettio, D. (2017). Acute promyelocytic leukemia with a cryptic insertion of RARA into PML on chromosome 15 due to uniparental isodisomy: A case report. Oncology Letters, 13, 4180-4184. https://doi.org/10.3892/ol.2017.5979 Other diagnostic methods RT-PCR Takes about 1-2 days Immunophenotyping Other tests Coagulation, PT, aPTT , CBC etc 05 Treatment Treatment Induction Maintenance All-Trans Retinoic Acid ATRA, ATRA + (ATRA), Arsenic Trioxide Chemo (ATO) Consolidation Side Effects ATRA + ATO Differentiation syndrome, chest pain, fever, fertility etc Recommendations Start treatment immediately upon suspicion of APL, even before genetic confirmation, to prevent hemorrhagic complications. There is a need for a quicker diagnostic technique with higher accuracy to provide better treatment options at the earliest. Perform bone marrow assessment to determine the response to ATRA. Classify patients into low, intermediate, or high risk based on the white blood cell (WBC). Peripheral blood smear and FISH for the fusion of PML/RARA should be expedited for rapid diagnosis of this time-sensitive disease. There are variant chromosomal aberrations are identified in up to 5% of APL cases including, t (11; 17)(q23; q21), t (5;17) (q35; q12-21), t (11; 17)(q13; q21) Conclusion APL, or acute promyelocytic leukemia, is a unique and extremely curable subtype of acute myeloid leukemia that is identified by the PML-RARA fusion gene and the t(15;17) translocation. With the development of specific drugs, such as arsenic trioxide (ATO) and all-trans retinoic acid (ATRA), the prognosis for APL has greatly improved, moving it from a disease with a high death rate to one with a high rate of cure. When APL is suspected, medication must be started right away in order to avoid potentially fatal consequences including coagulopathy. An important and crucial step in treating (APL) is diagnosis, requiring for a combination of genetic, morphological, cytochemical, and immunophenotypic investigations.  References Acute Promyelocytic Leukemia (APL): A Review of the Classic and Emerging Target Therapies towards Molecular Heterogeneity - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Chromosomal- translocation-between-chromosomes-15-and-17-Acute-promyelocytic-leukemia-is_fig1_368020769 [accessed 26 May, 2024] Amoth H, Perry AM. APL with PML::RARA. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/leukemiaAPL.html. Accessed May 27th, 2024. An In Vitro Model of Hematotoxicity: Differentiation of Bone Marrow–Derived Stem/Progenitor Cells into Hematopoietic Lineages and Evaluation of Lineage‐Specific Hematotoxicity - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Hierarchy-of-hematopoietic-cells-during-normal-differentiation-of-bone-marrow-derived_fig1_324848224 [accessed 27 May, 2024] Baljevic, M., Park, J. H., Stein, E., Douer, D., Altman, J. K., & Tallman, M. S. (2011). Curing All Patients with Acute Promyelocytic Leukemia: Are We There Yet? Hematology/Oncology Clinics of North America, 25(6), 1215–1233. https://doi.org/10.1016/j.hoc.2011.10.002 Cingam, S. R., & Koshy, N. V. (2023, June 26). Acute Promyelocytic Leukemia. StatPearls - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK459352/ Cytogenetic Techniques in Diagnosing Genetic Disorders - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/A-karyotype-of-a-normal-male-46-XY-Reproduced-courtesy-of-Human-Genome- Centre_fig1_221920029 [accessed 27 May, 2024] Ghiaur, A., Doran, C., Gaman, M. A., Ionescu, B., Tatic, A., Cirstea, M., Stancioaica, M. C., Hirjan, R., & Coriu, D. (2024). Acute Promyelocytic Leukemia: Review of Complications Related to All-Trans Retinoic Acid and Arsenic Trioxide Therapy. Cancers, 16(6), 1160. https://doi.org/10.3390/cancers16061160 Liquori, A., Ibañez, M., Sargas, C., Sanz, M., Barragán, E., & Cervera, J. (2020). Acute Promyelocytic Leukemia: A Constellation of Molecular Events around a Single PML-RARA Fusion Gene. Cancers, 12(3), 624. https://doi.org/10.3390/cancers12030624 Pagnano, K. B. B., Rego, E. M., Rohr, S., Chauffaille, M. D. L., Jacomo, R. H., Bittencourt, R.,... & Bernardo, W. (2014). Guidelines on the diagnosis and treatment for acute promyelocytic leukemia: associação brasileira de hematologia, hemoterapia e terapia celular guidelines project: associação médica brasileira-2013. Revista brasileira de hematologia e hemoterapia, 36, 71-92. Pandolfi P. P. (2001). Oncogenes and tumor suppressors in the molecular pathogenesis of acute promyelocytic leukemia. Human molecular genetics, 10(7), 769–775. https://doi.org/10.1093/hmg/10.7.769 Venci, A., Mazza, R., Spinelli, O., Di Schiena, L., & Bettio, D. (2017). Acute promyelocytic leukemia with a cryptic insertion of RARA into PML on chromosome 15 due to uniparental isodisomy: A case report. Oncology Letters, 13, 4180- 4184. https://doi.org/10.3892/ol.2017.5979 Wrede, J., Sundram, U., Kohler, S. et al. Fluorescence in situ hybridization investigation of cutaneous lesions in acute promyelocytic leukemia. Mod Pathol 18, 1569–1576 (2005). https://doi.org/10.1038/modpathol.3800465 Thank You Any Questions?