““ White blood / White blood / WeissesWeisses BlutBlut ““

Claudia MeyerClaudia MeyerEdendaleEdendale hospitalhospital

October 2007October 2007

Index patient: Index patient: Main complaintMain complaint

21 year old gentleman from 21 year old gentleman from EdendaleEdendaleMain complaint:Main complaint: Gingival bleeding and fatigue Gingival bleeding and fatigue for 7 monthsfor 7 monthsEpistaxisEpistaxis and dizziness for 2 weeksand dizziness for 2 weeksNo No haematuriahaematuria, no , no haematemesishaematemesis, no , no melaenamelaena, no , no haemoptysishaemoptysisPMH:PMH: HIV positive, CD 4 unknown, not on HAART. HIV positive, CD 4 unknown, not on HAART. No previous opportunistic infections, no previous No previous opportunistic infections, no previous malignancymalignancyDrugs:Drugs: Nil. Not on CoNil. Not on Co--trimoxazoletrimoxazole

History cont.History cont.Allergies:Allergies: Nil knownNil knownSocial:Social: NonNon--smoker. No ethanol use. Salesman. smoker. No ethanol use. Salesman. One sister from same parents, 8 halfOne sister from same parents, 8 half--siblings. siblings. No occupational exposure to radiation, No occupational exposure to radiation, pesticides, benzene.pesticides, benzene.Family History:Family History: Nil of note.Nil of note.Travel History:Travel History: Nil.Nil.Systemic enquiry:Systemic enquiry: No LOW, no night sweats or No LOW, no night sweats or fever. Decreased exercise tolerance. No other fever. Decreased exercise tolerance. No other respresp or or cvscvs S. No S. No ecchymosisecchymosis or jaundice. No or jaundice. No polyarthralgiapolyarthralgia, no photosensitivity., no photosensitivity.

Clinical examinationClinical examination

General :General : Pallor,Pallor, anictericanicteric, , acyanoticacyanotic, small , small mobile LN mobile LN submandibularsubmandibular, no , no oedemaoedema, no oral , no oral candidiasiscandidiasisNo No ecchymosisecchymosis, no skin nodules, no lesions , no skin nodules, no lesions suggestive of Kaposisuggestive of Kaposi’’s sarcomas sarcomaVital signs:Vital signs: ApyrexialApyrexial, , BP 99/48 mmHg, BP 99/48 mmHg, HR 112bpm,HR 112bpm, weight 57kgweight 57kgCVS:CVS: APPE, JVP APPE, JVP →→, tachycardia, regular, good , tachycardia, regular, good volume, apex volume, apex undisplacedundisplaced, , hyperdynamichyperdynamic, , heart sounds normal, 2/6 ESM at LSBheart sounds normal, 2/6 ESM at LSB

Clinical examinationClinical examination

RespResp:: RR 14, resonant, clear, RR 14, resonant, clear, bilatbilat b/sb/sAbdomen: soft, non-tender, no organomegalyNeuro: alert, orientated, no focal signs, no retinal haemorrhagesENT: no gingival hypertrophy, enlarged tonsils bilaterally, but not inflamed.Urine: No macroscopic or microscopic haematuria. No proteinuria. No urobilinogen.Ward Hb: 2 g/dl

Clinical assessmentClinical assessment21 year old salesman presents with symptoms 21 year old salesman presents with symptoms suggestive of a qualitative or quantitative suggestive of a qualitative or quantitative platelets defect and severe platelets defect and severe anaemiaanaemia, in the , in the absence of absence of palapablepalapable organomegalyorganomegaly..The patient is HIV positive, without prior The patient is HIV positive, without prior opportunistic infections.opportunistic infections.Differentials: Differentials: AplasticAplastic anaemiaanaemiaMalignancies: ALL, AML, lymphomaMalignancies: ALL, AML, lymphomaMyelodysplasiaMyelodysplasiaOpportunistic infectionsOpportunistic infections

InvestigationsInvestigationsFBCFBC: : HB 3.2 g/dl; MCV 87 fl; MCH 32.2 pg; HB 3.2 g/dl; MCV 87 fl; MCH 32.2 pg; WCC 1.25; abs WCC 1.25; abs neutrneutr 0.43, abs lymph 0.58; 0.43, abs lymph 0.58; mono 0.02; PLT 20, MPL 7.8 flmono 0.02; PLT 20, MPL 7.8 flCorrected Corrected ReticulocyteReticulocyte count : count : 0.77%0.77%U&E, CMP: normal, LDH: 275 iu/l, Uric acid: 0,29 mmol/lLFT:LFT: TP 95 TP 95 g/lg/l,, alb 21 alb 21 g/lg/l,, rest normalCoagulation: 1.22; APTT 23.7 secs, control 28,9 secs, D-Dimer 1000 ng/ml, Fibrinogen 2,0 g/lCD4 count: CD4 count: 268 cells/268 cells/ulul

Investigations cont.Investigations cont.Smear:Smear: pancytopeniapancytopenia,, anisocytosisanisocytosis 2+, 2+, occasional occasional promyelocytespromyelocytes notednotedUrgent BMAT and TrephineUrgent BMAT and Trephine: : Acute Acute promyelocyticpromyelocytic leukemia, trephine leukemia, trephine demonstrates demonstrates neoplasticneoplastic infiltration.infiltration.PCR, PCR, cytogeneticcytogenetic tests and flow tests and flow cytometrycytometrypendingpending..Ultrasound abdomen: no Ultrasound abdomen: no organomegalyorganomegaly, no , no lymphadenopathylymphadenopathy, normal , normal CXR: normalCXR: normalBlood culture, uBlood culture, u--MCS: no growthMCS: no growth

Acute Acute myelocyticmyelocytic leukemialeukemia

Acute Acute myelocyticmyelocytic leukemialeukemiaHeterogeneous group of diseasesHeterogeneous group of diseasesClonalClonal disorder of disorder of haematopoietichaematopoietic progenitor progenitor cells which lose the ability to differentiate cells which lose the ability to differentiate normally and to respond to normal regulators normally and to respond to normal regulators of proliferation.of proliferation.Incidence of AML 3,6 per 100 000 per year, Incidence of AML 3,6 per 100 000 per year, increases with age. Median age at increases with age. Median age at presentation at 70 years.presentation at 70 years.Acute Acute promyelocyticpromyelocytic leukemia is a subtype of leukemia is a subtype of AML, median age of presentation 25 years.AML, median age of presentation 25 years.

EtiologyEtiologyHeredity:Heredity: Somatic cell Somatic cell aneuploidyaneuploidy egeg. Trisomy21; . Trisomy21; Excessive chromosome fragility Excessive chromosome fragility egeg Bloom SBloom SIonisingIonising radiation:radiation: ↑↑ Incidence AML 5Incidence AML 5--7 yrs 7 yrs after atomic bomb explosion. after atomic bomb explosion. Chemicals:Chemicals: Benzene, smoking, petroleum Benzene, smoking, petroleum products, paint, ethylene oxide, pesticides.products, paint, ethylene oxide, pesticides.Drugs:Drugs: 1. 1. AlkylatingAlkylating agentagent--associated associated leukemiasleukemias44--6 yrs after exposure (6 yrs after exposure (chrchr 5; 7); 2. 5; 7); 2. TopoTopo--isomeraseisomerase II inhibitorII inhibitor--associated associated leukemiasleukemiasoccur 1occur 1--3 yrs after exposure (3 yrs after exposure (chrchr 11; t 15:17; 11; t 15:17; t 8:21); 3. t 8:21); 3. ChloramphenicolChloramphenicol, , chloroquinechloroquine →→BM failure that may evolve into AMLBM failure that may evolve into AML

HaematoHaemato--poiesispoiesis

NeutrophilNeutrophildevelopmentdevelopment

MultipotentMultipotent stem cellstem cell↓↓

Common myeloid progenitor cellCommon myeloid progenitor cell↓↓

GM GM CommitedCommited precursor cell precursor cell ↓↓

Granulocytic progenitor cell ( lineage committed cells )Granulocytic progenitor cell ( lineage committed cells )↓↓

MyeloblastMyeloblast →→↓↓

←← PromyelocytePromyelocyte↓↓

MyelocyteMyelocyte↓↓

MetamyelocyteMetamyelocyte↓↓

Band form Band form →→↓↓

NeutrophilNeutrophil

ClassificationClassificationFrench-American-British Classification (1985)8 major subtypes according to morphology & cytochemisty. Required > 30% myeloblasts in BM for diagnosis.M0 Minimally differentiated leukemiaM1 Myeloblastic leukemia without maturationM2 Myeloblastic leukemia with maturationM3 Hypergranular promyelocytic leukemiaM4 Myelomonocytic leukemiaM5 Monocytic leukemiaM6 ErythroleukemiaM7 Megakaryoblastic leukemia

WHO ClassificationWHO ClassificationRequires presence of > 20% Requires presence of > 20% myeloblastsmyeloblasts in blood in blood and/or BM. Incorporates molecular & and/or BM. Incorporates molecular & cytogeneticcytogenetic, , morphologic (multimorphologic (multi--lineage lineage dysplasiadysplasia ), clinical ), clinical features ( prior features ( prior haematologichaematologic disorder ).disorder ).

-- Recurrent translocations: t(8;21)(q22;q22), Recurrent translocations: t(8;21)(q22;q22), t(15;17)(q22;q22), t(16;16)(p13;q22). t(v:11q23)t(15;17)(q22;q22), t(16;16)(p13;q22). t(v:11q23)

-- MultiMulti--lineage lineage dysplasiadysplasia-- Therapy related AML & Therapy related AML & myelodysplasticmyelodysplastic SS-- Not otherwise Not otherwise categorisedcategorised-- Acute Acute myelomonocyticmyelomonocytic LL-- Acute leukemia of ambiguous lineageAcute leukemia of ambiguous lineage-- BilinealBilineal acute Lacute L

LeukemogenesisLeukemogenesis of AMLof AMLTwo-hit model:Class 1 genetic damage: results in constitutive activation of cell surface receptors ( eg RAS ), or receptor tyrosine kinases ( eg FLT 3, c-KIT ) →clonal expansion Class 2 genetic damage: results in formation of fusion genes that block myeloid differentiation.

FAB Classification & associated genetic FAB Classification & associated genetic abnormalitiesabnormalities

LeukemogenesisLeukemogenesis of M3of M3RARsRARs are nuclear receptors that act as are nuclear receptors that act as ligandligand--dependent dependent transciptiontransciption factorsfactors

↓↓t ( 15;17) ( q22;q12)t ( 15;17) ( q22;q12)

↓↓PMLPML--RARRARαα fusion protein binds fusion protein binds retinoid acid response elementsretinoid acid response elements

↓↓suppresses gene transcription suppresses gene transcription

and blocks differentiation of the celland blocks differentiation of the cell↓↓

differentiation at differentiation at promyelocytepromyelocytestage arrestedstage arrested

Clinical presentationClinical presentationSymptoms related to pancytopaenia: easy fatigue, infections, hemorrhagic findingsConstitutional Sx: LOA, LOW, feverPallor, fever, infection, hemorrhage including retinal hemorrhagesHepatosplenomegaly, lymphadenopathyInfiltration of gingivae, skin, soft tissues or meninges ( monocytic subtype M4 and M5)Granulocytic sarcoma ( chloroma ): mass consisting of leukemic cells in soft tissues, breast, uterus , ovary, GIT, lung or other organs → associated with t(8;21)

Clinical presentation: M3Clinical presentation: M3APL can be complicated by DIC ( at diagnosis or initiation of chemotherapy). Mechanism: 1. Tissue factor → forms a complex with F VII to activate F X and IX2. Cancer procoagulant, which activates F X3. ↑ Annexin R expression on surface of leukemic promyelocyes→ annexin II R binds plasminogen & tissue plasminogen activator →this results in increased plasmin formationThrombocytopenia as well as platelet Thrombocytopenia as well as platelet dysfunctiondysfunction

Diagnosis

BM aspirate & trephine biopsyBM aspirate & trephine biopsyCytogeneticCytogenetic: RT: RT--PCR for AML 1PCR for AML 1--ETO and CBFBETO and CBFB--MYH 11 in nonMYH 11 in non--APL and PMLAPL and PML--RARA in suspected RARA in suspected APL, FISH in selected casesAPL, FISH in selected casesCytochemistryCytochemistry ( MPO or Sudan ( MPO or Sudan Black, combined esterase )Black, combined esterase )ImmunophenotypingImmunophenotyping for specific for specific intracellular & surface antigensintracellular & surface antigens

Treatment of AML: OverviewInduction Rx : age

younger > 60 – 65 yrs↓↓ ↓↓

““ 3 + 7 3 + 7 ““ Standard Rx orStandard Rx or↓↓ investigational Rxinvestigational Rx

PostremissionPostremission RxRx: or supportive Rx: or supportive Rxrisk stratifyrisk stratify↓↓ ↓↓ ↓↓

favourablefavourable intermedintermed averse riskaverse risk(good) (standard) (poor)(good) (standard) (poor)

↓↓ ↓↓ ↓↓cytarabinecytarabine cytarabinecytarabine allogenicallogenic tplttplt

or new drugsor new drugs

Treatment of AMLInduction therapy: Aim is complete remission: BM < 5% blasts, neutrophils > 1000, PLT > 100 0003 + 7 combination “→ daunorubicin IVI 3 days, cytarabin IVI for 7 - 10 daysPostremissiontherapy: Risk stratify !Cytogeneticabnormalities

Elderly AML patientsAssociated with:Associated with:1. more adverse 1. more adverse cytogeneticscytogenetics2. 2. overexpressionoverexpression of MDR 1 gene encoding a drug of MDR 1 gene encoding a drug efflux pump pefflux pump p--glycoproteinglycoprotein3. 3. ↑↑ incidence of prior incidence of prior myelodysplasiamyelodysplasia4. generally do not tolerate chemotherapy as well as 4. generally do not tolerate chemotherapy as well as younger patientsyounger patientsOptions: Options: A) Standard treatment A) Standard treatment B) Palliative B) Palliative chemoRchemoR: low: low--dose dose cytarabinecytarabineC ) investigational treatment (clinical trial )C ) investigational treatment (clinical trial )D ) supportive care ( very poor performance D ) supportive care ( very poor performance

status, status, abnabn organ Function, > 80 yrs old )organ Function, > 80 yrs old )

General measure & supportive careGeneral measure & supportive careHyperleukocytosis: leucopheresis if Sx ( but not in PML)Tumour lysis S: hydration, allopurinol, monitor I + O, recombinant urate oxidase(rasburicase) Platelet transfusionPacked cell transfusionAnti-infective RxUse of prophylactic AB controversial

HaematopoieticHaematopoietic growth factorsgrowth factors

G-CSF given after completion of chemotherapy → reduce duration of neutropenia and decrease AB use, but no effect on rates of remission or overall survival.“ Priming effect “: G-CSF could stimulate leukaemic cells to proliferate, which could increase their susceptibility to chemotherapy→→ reduction in relapse and reduction in relapse and improved survival for patients with standardimproved survival for patients with standard--risk AML.risk AML.

Treatment M3: ATRAATRA as soon as ATRA as soon as DxDx of APL suspected and of APL suspected and given for prolonged time ( 21given for prolonged time ( 21--60days)60days)

Treatment M3Retinoic acid syndrome:Retinoic acid syndrome: ATRA → diff. of APL cells → cytokine release & surface adhesion molecule modulation → capillary leak & fluid retention as well as organ infiltration by leukaemic cells.Sx/Tx: cough, dyspnoea, fever, pulminfiltrates, pleural & pericardial effusion, oedema, Lab: ↑ WBCManagement: stop ATRA temporarily, dexamehtasone 10mg bd IVI; prophylactic steroids with induction Rx – benefit uncertain.

Treatment M3Induction: Induction: -- ATRAATRA-- daunorubicin & cytarabin IVIConsolidation:Consolidation: 22--3 3 anthracyclineanthracycline-- based based courses, ATRAcourses, ATRAMaintenance therapyMaintenance therapy: ATRA: ATRACoagulopathyCoagulopathy: : 1. Keep PLT > 50 until remission. 2. Keep coagulation times normal with FFPs3. Anti-fibrinolytic agents if life threatening hemorrhage

Patient progressInitial resuscitation with packed cells, developed massive epistaxis 4 days later, hence received a total of 8 units packed cells and 1 pool of platelets over the next 10 days Cyclokapron, H2 receptor antagonist, Piperacillin-Tazobactam and GentamycinNo isolation facilities were available at EDH

Patient progressWhilst awaiting transfer to IALCH: ATRA 40mg bd, Prednisone 30mg dly, Omeprazole 20mg dly, CyclokapronThe patient is currently undergoing induction therapy at IALCH with ATRA, daunorubicin & cytarabin.

ReferencesReferencesEsteyEstey E. AML. Lancet 2006; 368:1894 E. AML. Lancet 2006; 368:1894 -- 9090LowenbergLowenberg G. AML. NEJM 1999; 341: 1051 G. AML. NEJM 1999; 341: 1051 -- 10591059British Committee for Standards in British Committee for Standards in HaematologyHaematology. . British Journal of British Journal of HaematologyHaematology 2006; 135: 450 2006; 135: 450 -- 474474UpToDateUpToDateSchifferSchiffer C. C. HematopoieticHematopoietic Growth factors and AML. Growth factors and AML. NEJM 2003; 349: 727 NEJM 2003; 349: 727 -- 729729LowenbergLowenberg B. B. EffecyEffecy of Priming with Gof Priming with G--CSF on the CSF on the outcome of chemotherapy for AML. NEJM 2003; 349: outcome of chemotherapy for AML. NEJM 2003; 349: 743 743 -- 5252MenellMenell J. J. AnnexinAnnexin II and Bleeding in acute II and Bleeding in acute promyelocyticpromyelocytic leukemia,. NEJM 1999; 340: 994 leukemia,. NEJM 1999; 340: 994 –– 10041004

References cont.References cont.

PedersenPedersen--BjergaardBjergaard. Insights into . Insights into leukemogenesisleukemogenesisfrom therapyfrom therapy--related leukemia. NEJM 2005. 352: related leukemia. NEJM 2005. 352: 15911591--15941594McPheeMcPhee S. S. PathophysiologyPathophysiology of disease. Lange. Fifth of disease. Lange. Fifth edition p 116edition p 116KaushanskyKaushansky K. LineageK. Lineage--Specific Specific HematopoieticHematopoieticGrowth Factors. NEJM 2006;354:2034Growth Factors. NEJM 2006;354:2034--4545

Thank you !Thank you !

Molecular monitoringMolecular monitoring : RT: RT--PCR assay to PCR assay to detect detect minimal residual disease and relapse.minimal residual disease and relapse.Relapse:Relapse: ATRA + ATRA + chemoRxchemoRx followed by rescue followed by rescue using using allogenicallogenic stem cellsstem cells

Mechanism of chemotherapy

ATRA:ATRA: AllAll--trans retinoic acid ( trans retinoic acid ( tretinointretinoin): ): discussed previouslydiscussed previouslyCytarabineCytarabine: : inhibition of DNA polymeraseinhibition of DNA polymeraseDaunorubicinDaunorubicin: : AnthracyclineAnthracycline ABAB1. inhibits 1. inhibits topoisomerasetopoisomerase IIII2. High affinity binding to DNA through 2. High affinity binding to DNA through intercallationintercallation →→ results in blockade of DNA & results in blockade of DNA & RNA synthesisRNA synthesis3. Alteration in cell membrane transport3. Alteration in cell membrane transport4 Generation of oxygen free radicals that 4 Generation of oxygen free radicals that cause DNA strands to breakcause DNA strands to break

Topoisomerase II