APL - What a Blast!


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APL - What a Blast!

  1. 1. Acute Promyelocytic Leukemia MLSCI 430 Rabia Yousofi Tino Villatoro
  2. 2. Our Case Study  A 29-year-old male was admitted to ER looking pale with a rash on his extremities.  He has had a persistent nose bleed for the last 2 hours.  Lab Values: ◦ Hemoglobin 87 g/L ◦ Platelets 15 x 109/L ◦ WBC 38 x 109/L ◦ PT (INR) 5.0 ◦ PTT 62 seconds ◦ Fibrinogen 0.3 g/L
  3. 3. Further Lab Testing  A review of the peripheral blood smear revealed many abnormal cells.  A bone marrow aspiration and biopsy was later performed.  Samples were sent for flow cytometry, cytogenetics, and the molecular oncology lab.  A diagnosis of Acute Promyelocytic Leukemia (APL) was made on the basis of the tests performed.
  4. 4. What is Acute Promyelocytic Leukemia?  APL is characterized by the accumulation of blasts that are blocked at the promyelocytic stage of differentiation. http://www.pnas.org/content/102/20/7174/F6.large.jpg
  5. 5. WHO vs. FAB  Acute promyelocytic leukemia falls under the old FAB classification as AML FAB M3 and M3v (for the microgranular variant).  Under the new WHO classification, APL falls under the category AML with recurrent genetic abnormalities.  This new classification recognizes the molecular/genetic feature of APL, namely the balanced translocation of chromosome 15 and 17.
  6. 6. PML-RARA: t(15;17)  The t(15;17) is characteristic and virtually diagnostic of APL.  This translocation results in the fusion of the PML gene on chromosome 15q22 and the RARA (Retinoic Acid Receptor A) on chromosome 17q21.  Expression of the PML-RARA protein results in a block in differentiation at the promyelocyte stage by suppressing RARA target genes.
  7. 7. Variant t(15;17)  There is a common breakpoint within intron 2 of the RARA gene and three breakpoints within the PML gene which results in the formation of three variants.  These breakpoints are: ◦ Intron 6 (bcr1; 55% of cases) ◦ Exon 6 (bcr2; 5% of cases) ◦ Intron 3 (bcr3; 40% of cases)  Bcr3 is associated with M3v microgranular form. It has a higher incidence of DIC and a higher leukocyte count.
  8. 8. Variant APL Translocations  Other variant translocations may occur involving chromosome 17 that lead to APL: ◦ t(11;17)(q23;q21) ◦ t(5;17)(q35;q21) ◦ t(11;17)(q13;q21) ◦ der(17) (17q21.3-q23) http://www.pathguy.com/lectures/m3.jpg
  9. 9. Variant Translocations  t(11;17) (q23;q21) ◦ Most common and intensively studied variant ◦ Fuses the PLZF gene (promyelocytic leukemia zinc finger) with RARA resulting in the expression of a PLZF-RARA protein. ◦ Falls into an unusual morphologic spectrum of APL, with features intermediate between M2 (AML with some maturation) and M3 (APL). ◦ Important to recognize, as this translocation is insensitive to ATRA
  10. 10. Variant Translocations  t(5;17) (q35;q21) ◦ Second-most common variant ◦ This variant translocates the nucleophosmin gene on 5q35 into the RARa locus on 17q21 ◦ Nucleophosmin is a nucleolar phosphoprotein that plays a role in ribosomal RNA assembly; it also has chaperoning activities, as well as nuclease activity. ◦ The phenotype is identical to APL M3 ◦ In-vitro studies have shown that promyelocytes of t(5;17) are still sensitive to ATRA, and this has been shown in one case study as well.
  11. 11. Variant Translocations  t(11;17)(q13;q21) ◦ This is a rare APL variant ◦ Blood smear and bone marrow specimens show a predominance of promyelocytes and dysplastic maturing neutrophils. ◦ This variant is still sensitive to ATRA  der(17) (17q21.3-q23) ◦ Morphologically similar to AML M1 (AML with minimal differentiation) with a minority of marrow blasts showing morphologic evidence for the M3v microgranular variant of APL. ◦ Shows no response to ATRA
  12. 12. Cytogenetic Diagnosis  Cytogenetic studies can reveal the abnormal karyotype in APL.  Several banding techniques are available, including giemsa-trypsin banding, r- banding, and c-banding.  Metaphase chromosomes are treated with trypsin and stained with Giemsa. This creates a unique banding pattern for each chromosome.
  13. 13. http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed&part=A1548&rendertype=figure&id=A1557 http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed&part=A1548&rendertype=figure&id=A1551
  14. 14. Fluorescent In-Situ Hybridization http://en.wikipedia.org/wiki/File:FISH_%28Fluorescent_In_Situ_Hybridization%29.jpg
  15. 15. Minimal Residual Disease  Quantitative reverse transcriptase PCR can be used to detect the PML-RARA transcript.  Patients with a higher transcript level tend to have a worse prognosis.  Treatment and remission can also be monitored using real-time quantitative RT- PCR.
  16. 16. Typical Features of Blast Cells  Myeloblast  Monoblast  Proerythroblast  Megakaryoblast  Lymphoblast
  17. 17. Myeloblast  < 1% of the normal bone marrow, not observed in normal blood  Vary in size, but are usually large  Nucleus is delicate, large, round or Sl oval, with prominent nucleoli. Stain purplish red with Wright stain. Chromatin stains evenly  Small to moderate amount of bluish nongranular cytoplasm  Three major types: Type I, II, and III
  18. 18. Type I  Fine nuclear chromatin  2 to 4 distinct nucleoli  Moderate rim of pale to basophilic cytoplasm  Without azurophilic granules Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 8).
  19. 19. Type II and III  Type II ◦ Nuclear and cytoplasmic features are similar to type I ◦ Delicate azurophilic granules in the cytoplasm (up to 20)  Type III ◦ Numerous azurophilic granules in the cytoplasm Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 9). http://www.pathologyoutlines.com/leukemia.html
  20. 20. Auer Rods  One characteristic feature of myeloblasts in AML ◦ Presence of Auer rods with abnormal azurophilic granules ◦ (60% - 70% of all cases) ◦ (faggot cells) Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 10).
  21. 21. Monoblast  Derived from myelocytic-monocytic progenitor cells in BM  Round, sometimes folded, large early nucleus with 1 or 2 nucleoli  Finely dispersed linear chromatin  Small indentation (nuclear creases)  Basophilic cytoplasm with no granules (sometimes fine granules & occasional vacuoles)
  22. 22. Erythroblast  0% - 1% in normal bone marrow of adults  Round nucleus, Sl condensed nuclear chromatin  Variable prominent nucleoli  Moderate amount of deeply basophilic cytoplasm.
  23. 23. Megakaryoblast  Moderately large cell with 1 or 2 nuclei (round nucleus)  Nucleus begins to indent with lobes start to form and nucleus increases in size  Nucleoli often demonstrable  Non-granular cytoplasm, may have blunt pseudopods or blebs, stain bluish  In AML, very significantly in appearance from one case to another
  24. 24. Promyelocyte  Primary granules (azurophilic or dark blue), no secondary granules  1% - 5% in normal bone marrow, not seen in normal blood  Its size varies (may exceed 20µm)  Nucleus is round and large in relation to the cytoplasm  Chromatin is sl courser than myeloblast  Nucleoli may be visible (often indistinct)  Cytoplasm is dark blue with a relatively light area adjacent to the nucleus http://www.healthsystem.virginia.edu/internet/hematology/hessidb/normal-hematopoietic-cells.cfm
  25. 25. Neoplastic Promyelocyte (Blast Equivalent)  Eccentric, often folded and lobulated nucleus  Sl condensed nuclear chromatin  Intense cytoplasmic granulaity  An apparent Golgi zone Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 10).
  26. 26. Cytochemical Stains  Since the early 20th century, cytochemical staining of cells has been a useful tool in differentiating hematopoietic diseases.  Smears and imprints made from bone marrow, lymph nodes, spleen, or peripheral blood are preferred. ◦ In enzymatic techniques, fresh smears are used to ensure optimal enzyme activity  Certain elements may be inhibited during the fixation of smears and imprints
  27. 27. Myeloperoxidase (MPX/MPO)  The proxidase enzyme reacts with H2O2 & release O2, which oxidizes the indicator dye and produce orange-brown granules in the cells (3-amino-9- erythrocarbazol)  Enzyme MPX is found in the 1o granules of granulocytes, neutrophils and precursors (from the promyelocyte stage on) & eosinophils  Monocytes may be weakly pos  Leukemic myeloblasts are usually pos and Auer rods stain very strongly  Used for differentiating AML (+) from ALL (-)  Normal bone marrow smear <5 days old used for control slides (promyelocyte - neutrophils)
  28. 28. http://www.aquinaspathology.com/images/sp_MyeloperoxidaseStainAcut.jpg http://www.dfhcc.harvard.edu/core-facilities/specialized-histopathology-services-pathology/services/
  29. 29. Non-Specific Esterase (NSE)  Nonspecific esterase liberates alpha-naphthyl from the substrate alpha-naphthyl acetate. Alpha-naphthyl is couples with the dye molecule to form dark reddish-brown granules  Monocytes, monblasts, macrophages, histiocytes, megakaryocytes and some carcinomas are NSE pos  Abnormal erythroblasts are strongly pos  Lymphocytes are neg or may show dot positivity
  30. 30. NSE continued  Used for differentiating myelomonocytic and monocytic leukemia (+) from granulocytic leukemia (-)  Monocyte NSE are fluoride sensitive  Peripheral smear with appreciable # of monocytes or a normal BM smear used for control slides http://www.healthsystem.virginia.edu/internet/hematology/hessedd/malignanthematologicdisorders/leukemias/aml-m4.cfm
  31. 31. Periodic Acid Schiff (PAS)  Periodic acid oxidizes glycogen, mucoproteins, and other high-molecular weight carbohydrates to aldehydes.  Aldehydes react with colorless Schiff reagent, staining them a bright red-pink  Megakaryocytes and platelets stain strongly pos  Normoblasts will stain Pos  Lymphoblasts in ALL show course and granular (block) positivity
  32. 32. PAS Continued  Myeloblasts are Neg  Aids in diagnosis of ALL, erythroleukemia, and megakaryoblastic leukemia  Normal bone marrow smear used for control slides http://www.pathologyoutlines.com/leukemia.html
  33. 33. What about those coag tests?  The coagulation tests performed were very abnormal and are suggestive of disseminated intravascular coagulation (DIC).  80% of APL cases present with hemorrhagic manifestations http://www.hoslink.com/haematology/purp.jpg
  34. 34. Pathophysiology of DIC  A widespread systemic activation of coagulation resulting in diffuse fibrin deposition in the microvasculature.  This can lead to multi-organ dysfunction; red blood cell shearing; consumption of coagulation factors and platelets; and bleeding. http://www.pathologystudent.com/wp-content/uploads/2009/07/DIC_With_Microangiopathic_Hemolytic_Anemia_301920983.jpg
  35. 35. The APL connection  When promyelocytes release the contents of their primary granules, their pro- coagulant activity initiates DIC.  Aside from increased expression of procoagulant activity, there is also an activation of primary fibrinolysis and inflammation.
  36. 36. DIC and the lab  Consumption of coagulation factors leads to prolonged PT and PTT, and a decrease in fibrinogen. ◦ Factors consumed: fibrinogen, factor V, factor VIII and factor XIII.  Fibrinolysis of cross-linked fibrin leads to the formation of fibrin degradation products, notably D-Dimer.
  37. 37. Fibrinogen  The thrombin clotting time of dilute plasma is inversely proportional to the concentration of fibrinogen.  The method for measuring fibrinogen involves testing dilutions of patient and control plasma with excess thrombin.  Results are calculated using a standard curve. http://labsystems.roche.com/content/products/sta_r/benefits.html
  38. 38. Diagnosing DIC  Aside from the prolongation of the PT and PTT, the decreased fibrinogen, and CBC results, there are a number of tests used to diagnose DIC including: ◦ Antithrombin Levels ◦ D-Dimer ◦ Prothrombin Fragment F1.2
  39. 39. Antithrombin Assay  Antithrombin III will complex with thrombin and activated factor X, resulting in diminished plasma levels in DIC.  Micro-latex particles are coated with antibodies against antithrombin. Light is passed through the cuvette of a wavelength much greater than the diameter of the particles.  An increase in absorbance at 570 nm is directly proportional to the concentration of antithrombin.
  40. 40. Antithrombin Levels  Antithrombin levels provide an indirect measurement of thrombin activation.  Antithrombin levels may be decreased due to a hereditary deficiency or one of many acquired deficiencies including: ◦ DIC ◦ nephrotic syndrome ◦ liver disease ◦ oral contraceptive use, ◦ post-surgery ◦ prolonged heparin therapy. http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=coffeebrk&part=A22
  41. 41. D-Dimer Assay  Plasmin will lyse stabilized fibrin clots and form D-dimers as well as other fibrin degradation products.  Micro-latex particles are coated with monoclonal antibodies against human D- dimer. Light is passed through the cuvette of a wavelength much greater than the diameter of the particles.  An increase in absorbance at 570 nm is directly proportional to the concentration of D-dimer.
  42. 42. http://www.vet.uga.edu/VPP/clerk/ayoob/fig02_adj.jpg
  43. 43. D-Dimer Levels  D-dimer levels are elevated in situations where active thrombosis is occurring, notably: ◦ DIC ◦ Deep Vein Thrombosis ◦ Pulmonary Embolism  D-dimer levels will not be increased in primary fibrinolysis (only cross-linked fibrin will lead to D-dimer formation)  The assay has a great negative predictive value, but a positive result must correlate with other lab values in the diagnosis of DIC
  44. 44. Prothrombin Fragment F1.2  The conversion of prothrombin to thrombin involves the cleavage of a small molecule off the parent molecule.  The measurement of this fragment reflects thrombin activation and active thrombosis.  One of the methods of measurement employs a monoclonal antibody against the fragment, in an ELISA sandwich assay.
  45. 45. Prothombin F1.2 Levels  Increased levels reflect thrombin activation and active thrombosis, which may occur in DIC or other hypercoagulable states.  The use of prothrombin fragement F1.2 is not often used in the diagnosis of DIC.  Insufficient experience with the measurement of prothrombin F1.2 may limit it’s usefulness relative to other diagnostic tests for DIC.
  46. 46. Treatment: All-Trans Retinoic Acid (ATRA)  Considered as a first line therapeutic drug in the treatment for APLs  Acts by promoting terminal differentiation of the abnormal promyelocytes to mature neutrophils  Highly effective in induction of complete remission  Safe, convenient and cheap  Prevent the fatal bleeding caused by DIC, reduction of early mortality (major advantage)  ATRA is an isomer of retinoic acid (RA)
  47. 47. Retinoic Acid  Retinoic acids (RA) are signalling molecules that play important roles during embryonic development, also influence physiological functions like organogenesis, organ homeostasis and growth, differentiation or death of adult cells.  RA isomers are a group of active metabolites of vitamin A and their physiological effects are mediated through two families of RA receptors: RARs and retinoid X receptors (RXRs).
  48. 48. Retinoic Acid Continued  There are 3 members in each receptor family, encoded by different genes, namely RARα, β, and γ, and RXRα, β, and γ  Promotes myeloid differentiation http://www.cisreg.ca/cgi-bin/tfe/articles.pl?tfid=337
  49. 49. Leukemogenesis of APL  The PML–RARα chimeric protein acts as a dominant negative mutant over wild-type RARα by forming a homodimer and prevents activation of key RA target genes  The leukemia-specific fusion proteins display a higher avidity for corepressors of RARα.  As a result, the RARα/RXR pathway necessary to the granulocytic differentiation is abolished. http://www.bioscience.org/2009/v14/af/3333/figures.htm
  50. 50. Leukemogenesis of APL • PML-RARα forms homodimer through the coiled-coil motif of PML and competes with RARα for binding to RARE of target genes
  51. 51. Mechanism of ATRA  Pharmacologic dosage of ATRA directly modulates PML-RARα and its interaction with the nuclear receptor co-repressor complex ◦ Restores the wild-type RARα/RXR regulatory pathway and induces the transcriptional expression of downstream genes  ATRA can induce degradation of the PML– RARα oncoprotein, leading to activation of repressed target genes
  52. 52. Mechanism of ATRA http://rstb.royalsocietypublishing.org/content/362/1482/959.full
  53. 53. Modulation of the interaction of the receptor with CoR or CoA  At physiological concentration (0.01 mM), ATRA can dissociate CoR from wild-type RARα/RXRα and recruit CoA for transcriptional activation  PML-RARα is less sensitive to ligand-induced modulation, 0.1 ~ 1 mM (pharmacological concentrations) of ATRA is needed http://www.bioscience.org/2009/v14/af/3333/figures.htm
  54. 54. Limitations of ATRA  Patients with another translocation involving RARα that results in expression of the PLZF-RAR α protein, are insensitive to ATRA and arsenic trioxide (CT)  ATRA will ↑ the WBC count and cause leukocytosis rapidly which may lead to lethal consequences  Causes retinoic acid syndrome  Long term use may induce ATRA-resistance  1/3 to 1/2 of patients still relapsed probably due to a selection of clones resistant to ATRA (Relapse is the major subject of concern at present)
  55. 55. Combining ATRA with Chemotherapy  ATRA exerted its effect by inducing terminal myeloid differentiation, but could not prevent the occurrence of malignant transformation in myeloid progenitor cells  The combination of ATRA with a chemotherapeutic agent yields a higher CR rate and a longer overall survival  Arsenic Trioxide (ATO) combined with ATRA further improved the 5-year overall survival
  56. 56. Combining ATRA with Chemotherapy http://rstb.royalsocietypublishing.org/content/362/1482/959/F2.large.jpg
  57. 57. References  Bench AJ, Erber WN, Scott MA. Molecular genetic analysis of haematological malignancies: I. Acute leukaemias and myeloproliferative disorders. Clin Lab Haematol 2005 Jun;27(3):148-71.  Konoplev S, Bueso-Ramos CE. Advances in the pathologic diagnosis and biology of acute myeloid leukemia. Ann Diagn Pathol 2006 Feb;10(1):39-65.  Zhang JW, Wang JY, Chen SJ, Chen Z. Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells. J Biosci 2000 Sep;25(3):275-84.  Salih HR, Kiener PA. Alterations in Fas (CD 95/Apo-1) and Fas ligand (CD178) expression in acute promyelocytic leukemia during treatment with ATRA. Leuk Lymphoma 2004 Jan;45(1):55-9.  Chen ZX, Tao RF, Xia XM. The present status in all-trans retinoic acid (ATRA) treatment for acute promyelocytic leukemia patients: further understanding and comprehensive strategy are required in the future. Leuk Lymphoma 1992 Nov;8(4-5):247-52.  Zhou GB, Zhang J, Wang ZY, Chen SJ, Chen Z. Treatment of acute promyelocytic leukaemia with all-trans retinoic acid and arsenic trioxide: a paradigm of synergistic molecular targeting therapy. Philos Trans R Soc Lond B Biol Sci 2007 Jun 29;362(1482):959-71.
  58. 58. References Continued  Levi M, Jonge E, Poll T. Plasma and plasma components in the management of disseminated intravascular coagulation. Best Pract Res Clin Haematol 2006;19(1):127-42.  Bench AJ, Erber WN, Scott MA. Molecular genetic analysis of haematological malignancies: I. Acute leukaemias and myeloproliferative disorders. Clin Lab Haem 2005;27:148-71.  McCraw B. Diagnosing disseminated intravascular coagulopathy in acute promyelocytic leukemia. Clin J Oncol Nurs 2008;12(5):717-20.  Falanga A, Rickles FR. Pathogenesis and management of the bleeding diathesis in acute promyelocytic leukaemia. Best Pract Res Clin Haematol 2003;16(3):463-82.  Redner RL. Variations on a theme: the alternate translocations in APL. Leukemia 2002;16:1927-32.  Harmening DM. Clinical hematology and fundamentals of hemostasis. 5th ed. Philadelphia: F.A. Davis Company; 2009.  Rodak B F, Fritsma G A, Doig K. Hematology: clinical principle and applications. 3rd ed. Missouri: Saunders Elsevier; 2007.  Konoplev S, Bueso-Ramos CE. Advances in the pathologic diagnosis and biology of acute myeloid leukemia. Ann Diagn Pathol 2006 Feb;10(1):39-65.