1. A 27-year-old woman was admitted with fever and chest pain and was diagnosed with acute B-cell lymphoblastic leukemia (B-ALL) with the E2A-PBX1 fusion transcript based on morphological, immunophenotypic and cytogenetic analysis. 2. A 51-year-old man presented with cough, fever and petechiae and was diagnosed with acute myeloid leukemia (AML) with maturation based on the presence of Auer rods and the AML1-ETO fusion transcript. 3. A 53-year-old man underwent follow-up 10 years after treatment for follicular lymphoma and was later diagnosed with therapy-related myelodysplastic syndrome (

1. ESO Leukemia Lymphoma 2011Diagnosis and treatment in leukaemiaProf Jakob R Passweg

2. Case 1A 27-year-old woman is admitted with fever and thoracic pain. During the last week she experienced general fatigue and dyspnea during mild to moderate physical exertion.The physical examination, including cardiovascular and pulmonary examination was normal. There was no lymphadenopathy, splenomegalyor hepatomegaly

3. Hematology Lab

4. ScattergramPEROX: two major cell populations, large peroxidase positive cells (Neutr), and small to medium sized peroxidase negative cells (peroxidasenegative blasts and normal lymphocytes)BASO: nuclear shape. The predominant cell population is mononuclear

5. Peripheral blood smearsmallblastswithscantcytoplasm, condensednuclearchromatinandindistinctnucleoli, andlarger blastswithmoderate cytoplasm, smallcytoplasmicvacuolesand a distinctnucleoli. Noprimarygranulesor Auer rods35% blasts = acuteleukemia

6. BM Cytologyno marrow fragments but an adequate number of free marrow cells, predominance of small to medium sized blasts. Sudan Black staining: blast cells are negative. PAS staining: blast cells are negative. Morphologic diagnosis: acute peroxidase negative leukemia. Suggestive for an ALL.

7. BM HistologyGiemsa stain: densely infiltrated marrow, blasts monomorphicwith prominent nucleoli and narrow cytoplasm. Immunostaining, dim positive for TdT, strongly positive for CD10, and negative for CD20, CD3, CD117. (CD10 is shown) CD34 neg, (endothelial cells positive, (internal positive control)). The histological diagnosis is acute B-cell lymphoblastic leukemia

8. Flow CytometryBlasts: intracellular CD22, CD79a, IgM, and dimly TdT. Surface markers: CD19, CD10, and HLA-DR. Blasts:negative for immuno-MPO, CD34, surface IgM and for all myeloid and T-cell markers analyzed. The flowcytometric analysis demonstrates the B-lymphoid precursor cell phenotype of the acute leukemia. Diagnosis: acute B-cell ALL. Expression of cytoplasmic but not surface IgM allows to subclassify the leukemia as pre-B-cell ALL, without aberrant myeloid or T-cell markers

9. Classification according to EGILmature B Cell neoplasmsMature BCommon BPre-Bpro B AUL TdTCD19CD10cIgMsIgMIgG/IgD/ Kappa/Lambda

10. MolecularGeneticsMultiplex RT-PCR screening for 28 fusion transcripts. Eight multiplex RT-PCR amplifications, containing each, primers for a house keeping gene as well as primers for 4 to 5 fusions transcripts are included. additional gene product: suggesting the existence of an abnormal fusion transcript. split out: abnormal band, corresponding to the fusion transcript E2A/PBX1. Conventional cytogenetics: 46, XX, del(9)(q?12q32), del(11)(p?11.2),del(11)(q22q23) [6]46, XX [11] Definitive diagnosis Pre-B-ALL with E2A/PBX1 General Remarks The fusion transcript E2A/PBX1 corresponds to a genetic translocation type t(1;19)(q23;p13). This translocation has not been found with conventional cytogenetics, probably cryptic. However, other complex cytogenetic anomalies are found on chromosome 9 and 11. pre-B-cell ALL associated with t(1;19) and/or E2A/PBX1 fusion transcript. unfavorable prognosis

11. Treatment on GRAALL Steroid prephaseChemosensitivityevaluation d+8Double inductionR – Rituximabif CD20+R – HyperCVADvsconventionalMonitor MRD / B-cellreceptorgenerearrangement / flow / fusiontranscripts

12. Treatment on GRAALLTransplant in CR1 ?WBC > 30G/LCNS involvedB-ALL: CD10-; t(4;11); t(1;19)Complexcaryotype (>5); haploid ornear triploidCorticoresistanceChemoresistanceNo CR1 after induction

13. Case 2A 51-year-old gentleman, admitted for thoracic pain. During the last three weeks the patient presented non productive cough, fever, shortness of breath and petechiae.On physical examination: body temperature was 38.4 C° and moderate hepatomegaly could be observed. Conventional chest radiograph was normal, but on computed tomography two opacified pulmonary nodules, suggestive of fungal infection were observed in the lung periphery.

14. Hematology lab

15. ScattergramPerox: majority of rather small, peroxidase positive cells, distributed over three regions, the neutrophil (Neutr), monocyte (Mo) and eosinophil (Eo) corresponding to blast cells, and + residual neutrophils. Baso: The predominant cell population has a mononuclear nucleus (MNC). Only few cells a polymorphonuclear. This scattergram demonstrates, that the blast cells are strongly peroxidase positive -> AML.

16. Peripheral Bloodsmall blast with a thin rim of basophilic cytoplasm. The residual mature neutrophils show homogenous pink colored cytoplasm. Long Auer rods are found in the blast cells as well as in mature neutrophils. A blast proportion above 20%, with some of the blasts containing an Auer rod -> acute myeloid leukemia.

17. MarrowCytologyincreased cellularity , mostly large blasts abundant cytoplasm, prominent Golgi. Blasts with very large granules, (pseudo Chediak-Higashi). Mature neutrophils with prominent and sharp Auer rods. Most of the blast cells stain moderately to strongly for peroxidase. The Chediak-Higashi granules as well as the Auer rods show strong peroxidase positivity. The diagnosis based on morphology: AML with maturation (M2). Some of the morphologic characteristics are suggestive for an AML with t(8;21)(q22;q22)/AML1//ETO.

18. Histocellularity markedly increased (60-70%) blasts infiltration. pleomorphic blasts abundant cytoplasm. Blasts strongly positive for immunoperoxydase, CD34 CD117 dim. Histological diagnosis: acute myeloid leukemia (AML) with maturation

19. FlowBlasts, gated on CD45 dim, myeloid immunophenotype; positive for MPO, CD33, CD13, and express markers of immaturity (CD34, HLA-DR, CD117). Additionally they express a B-cell (CD19) and a NK-marker (CD56). The flowcytometric analysis confirms the myeloid phenotype of the acute leukemia. The aberrant marker expression of CD19 and CD56 are typically found in AML with translocation t(8;21) and/or AML1/ETO

20. DiagnosisMolecular genetics and Cytogenetics Using a Multiplex RT-PCR System for genotyping leukemia that screens for 28 different fusion transcripts, the blast cells are positive for the fusion transcript AML1/ETO. conventional cytogenetics, karyotype : 45, XX, -Y, t(8;21)(q22;q22) [13] Definitive Diagnosis Acute myeloid leukemia with t(8;21)(q22;q22); (AML1/ETO)

21. Treatment (HOVON SAKK 102 30/09)

22. Case 3A 53-year-old man seen for follow-up 10 years after autologous stem cell transplantation for follicular lymphoma. Follicular lymphoma of the tonsils. Cervical radiotherapy with 36 Gy. Relapse 5 years later follicular lymphoma IIIB treated by chemotherapy followed by autologous HSCT (CBV conditioning). Excellent general condition, no pain or fever, no weight loss and no night sweats. The physical examination, including cardiovascular examination was normal. Absence of palpable lymphnodes, spleen or liver.

23. Hem Lab values

24. Peripheral Blood Minor morphologic changes: platelets, with size variability, and neutrophils with granulation defect. Few pseudo-Pelgerneutrophils. No blast cells.

25. MarrowCytologyThe marrow aspirate is moderately hypercellular. Erythropoiesis without dysplastia, except for few erythroblasts with cytoplasmic vacuoles (). Myelopoiesis slightly increased with few immature forms. Megakaryocytes with dysplastic changes, hypolobular. On iron staining a few ringed sideroblasts.

26. Histologymarrow biopsy: moderately hypercellular marrow, restructured topography with clusters of erythroblasts and immature myeloid cells. Few abnormal megakaryocytes, with hypolobulated nuclei. Conclusions Suspicion of therapy related myelodysplastic syndrome (t-MDS).

27. Follow-up 6 monthslaterNo complaints, no treatment proposed6 months later: fatigue, no fever, no bleeding.

28. Hem Lab follow-up

29. Blood Smear @ FUEvident dysplastic changes of all three cell lines. RBC: remarkable anisocytosis and poikilocytosis. Platelets marked size variability and abnormal granulation Neutrophils have hypogranulation and nuclear hyposegmentation.

30. MarrowCytologyMarrow aspirate of poor quality, dysplastic changes of all three cell lines are evident. Erythroid hyperplasia, with important dyserythropoiesis. Increased number of megakaryocytes (small, hypolobulated nucleus). Few immature cells with abnormal chromatin pattern, but overall the number of blasts is below 5%. In iron staining, the proportion of ringed sideroblasts has increased, and is > 15% of all erythroblasts.

31. HistologyDensely hypercellular marrow, complete replacement of fat, trilineage hyperplasia. Marked proliferation of immature myeloid cells, abnormal localization, hypolobularmegakaryocytes, increased reticulin and secondary marrow fibrosis. Immunostains for CD34 shows some positive endothelial cells, few immature hemopoietic cells. The overall proportion of CD34-positive blasts is <5%.

32. GeneticsOnly 5 metaphases: 45, XY, add(5)(q11.2), dic(17;20)(p11.2;p112.2) [4]46, XY [1]Conclusions Therapy related myelodysplastic syndrome (t-MDS)refractory cytopenia with multilineage dysplasiawith ringed sideroblastsand cytogenetic anomalies

33. Another 6 months down theroadfatigue ++, petechiae ++* transfused

34. Cytogenetics12 metaphases: 45, XY, add(5)(q11.2), dic(17;20)(p11.2;p112.2) [1]45, idem, del(7)(q22) [1]45, idem, del(7)(q22),del(12)(p11.2),-21,+r [10] Definitive diagnosis Therapy related acute myeloid Leukemia (t-AML)Alkylating agent relatedwith complex cytogenetic anomalies

35. Comments Typical case of a therapy related MDS/AML, presenting initially with discrete morphologic changes, progressing than to a MDS type refractory cytopenia with multilineage dysplasia and evolving eventually to an AML. “alkylating agent” related MDS/AML, occuring 5-6 years following exposure to mutagenic agents. In the present case, mutagenicity can be due to the alkylating agents and to radiotherapy. Cytogenetically “alkylating agent” related MDS/AML present typically unbalanced translocations or deletions of chromosomal material. Alkylating related t-MDS/AML are associated with unfavorable outcome

36. Case 450-year old female patientProgressive fatigue since 4 monthsDiffuse upper abdominal pain No history of radiotherapy / drug exposure Slight splenomegalyblood count

37. Blood count

38. Peripheral blood smear

39. At this step what is the most probable diagnosis ?SeverebacterialInfectionMyeloproliferativedisorder (MPD)Chronicmyeloidleukemia (CML)MPD / MDSNone oftheabove

40. What is typical for CML?Leukocytosis Neutrophilia with pathologic left shiftIncreased number of myelocytes/metamyelocytesThrombocytosisAnisocytosis of plateletsIncreased number of eosinophilsIncreased number of basophils

41. What is absolutely necessary to confirm the diagnosisFlowcytometryBone marrow histology including immunohistchemistryCytogeneticsMolecular geneticsBone marrow cytology

42. Molecular genetic resultBcr/abltranscript positive b2a2Do we still needconventionalcytogenetics?

44. Why do we need morphology in CML patients ?Confirmation of the diagnosis of CMLDefine the stage of the diseaseTo complete the diagnosis To recognize possible atypical forms of CML or MPDFor treatment decision

45. Bone marrow cytology

46. Bone marrow histologyHematoxylin-eosin stainGömörri stain (silver stain)

47. Immunohistochemistry (anti CD34)40x20x

48. Stages of the CMLAtdiagnosisChronicphaseAcceleratedphaseBlast crisisMyeloid blast crisisLymphoid blast crisisDuringfollow-upRemissionHematologicalMorphologicalCytogeneticMajor MolecularCompleteMolecularChronicphaseAcceleratedphaseBlast crisis

49. Additional informationBonemarrowcytologyBlasts 1.5%, Basophils1%BonemarrowhistologyCD34 positive cells 2.5%Cytogenetics46,XX,t(9;22)(q34;q11)[20]

50. What is the stage of the disease at diagnosis?Myeloid blast crisisHematological remissionChronic phase Accelerated phaseNone of the above

51. Diagnostic criteria (WHO)for CML in accelerated phaseBlasts 10-19% in bloodand/orbonemarrow

52. Peripheralbloodbasophils> 20%

53. Persistent thrombocytopenia (<100x109/l) unrelatedtotherapy

54. orthrombocytosis (>1000x109/l) unresponsivetotherapy

55. Increasingspeensizeandincreasing WBC unresponsivetotherapy

56. CytogeneticevidenceofclonalevolutionDiagnostic criteria forCML-blast crisisBlasts ≥ 20% of PB and/or BMExtramedullary proliferation of blasts (myelosarcoma)Large aggregates and clusters of blasts in the BM-biopsyImmunophenotype of Blast crisis70% myeloid blasts, 30% lymphoblasts

57. Final diagnosisChronic myeloid leukemia (CML)t(9;22)(q43;q11)/BCR-ABL positivein chronic phase

58. How would you treat this patient?

59. Take home messagesThe diagnosis of CML depends on the presence of the characteristic t(9;22) or its respective BCR-ABL fusion transcript

60. Morphology of bone marrow is needed for definition of the stage of the disease

61. The classification in chronic, accelerated and blast phase is based on a combination of clinical, morphological and genetic findingsCase 579 year old man with absolute lymphocytosisAccidental findingPatient in good general healthClinical findingNo lymphadenopathy

62. No enlarged spleen or liverPeripheral blood count

63. Peripheral blood smear

64. Diverse mature lymphoid neoplasmsB-CLLHairy cell leukemiaSezary‘s SyndromeFollicular lymphomaT-LGL leukemiaProlymphocytic leukemia

65. Flow cytometric pattern of mature B-cell lymphoid neoplasms

66. What is the next step ?Minimal diagnostic programCoombs test, Immunoglobulin levels

67. Immunophenotyping from peripheral bloodResultsCoombs test negative

68. Immunoglobulin levels normal

69. no monoclonal fraction

70. Immunophenotyping (FACS)What is here the aim of immunophenotypingDistinction between neoplasm & reactive lymphocytesLight chain monoclonal lymphocytesDistinction between mature & precursor cell lymphoid neoplasmlymphoma / CLL: mature neoplasms ALL/ lymphoblastic NHL: Precursor cell neoplasmsDistinction between B-, T- & NK-cell neoplasmsDefining subtypes of lymphoid neoplasms

71. Immunophenotyping results

72. Algorithm of mature B-cell neoplasmsMonoclonal B-cells+-CD5++--CD23CD10othersCLLMCLFL

73. Complete result of the immunophenotypingKappa-monoclonalityCells are positive for CD19+, CD20+, CD5+, CD23+Negative or dim positive forsIgM dim, CD79b-, FMC7-, CD10-, CD103- Immunophenotypic conclusionMature B-cell neoplasm, Type B-CLLCLL score (matutes score) 5/5

74. Final diagnosisMature B-cell lymphoid neoplasia, type B-CLLRai 0, Binet Aasymptomatic No autoimmune phenomenaIn an elderly patient (79-years old)No genetic information available

75. What will be the treatment strategy?Watch and waitRegular follow-up (every 6 months)Constitutional symptomsOrganomegalyPeripheral blood countWhy is it acceptable to restrict diagnostics to a minimal program?elderly patient no intention of curative therapyasymptomatic patient no treatment required

76. Case 5b45 year old manCheck-up taking out insurance

77. No symptomsClinical findingsNo lymphadenopathy

78. No hepatomegaly, no splenomegaly

79. Five years previously, normal blood count and differential countFamily history of CLL - aunt, mothers side

80. Peripheral blood count

81. Confirmation of the B-CLLComplete ImmunophenotypeLymphocytes positive forCD19, CD20dim, CD23, CD5, kappa, sIgM dimLymphocytes negative for:FMC-7, CD79b, CD103, CD10

82. How to define management strategyYoung patient with B-CLLRai 0, Binet AConsiderations for management strategyAge of the patientAge - not an independent risk factor in CLLYounger patients are more likely to die from CLLStage of the disease: early stage is due to early stage with undefined prognosis or good prognosis CLL – smoldering diseaseWe need more information

83. Clinical prognostic factors in CLLSymptomatic CLL; B-symptomsFever, night sweat, weight lossDisease stageRai or BinetLymphocyte count> 50 x109/LDoubling time of absolute lymphocyte count< 12 months

84. Genetics -> Prognosisinterphase FISHGood prognosisIsolated del 13q14IntermediateTrisomy 12Normal karyotypePoor prognosisdel 11qdel17pdel 11q

85. Mutational status of IgVH genes in B-cell CLLTwo phases in B-cell developmentAntigen-independent phaseBone marrowGene rearrangementLymphoid follicle (follicular mantle)Unmutated B-cells naïve B-cellsAntigen-dependent phaseIn lymphoid follicle (germinal centre)Somatic hypermutation mutated B-cellsMemory cells

86. Biological surrogate markers for mutational statusMutational status not routinely availableCD38 expression on B-cellsPoor prognosis: positive ( 30%)No good correlation with mutational statusZAP70 expression on B-cellsPoor prognosis: positive (>20%)Better correlation

87. ZAP-70 expression by flow cytomteryZAP-70 positive B-cellsZAP-70 negative B-cells

88. Interpretation of the global situation and next stepsEarly stage, high risk B-CLL in a young patientCLL asymptomaticRai I / Binet A stage, lymphocyte countFISH del(11q); ZAP-70 positive, CD38 positiveWatch and wait as long as asymptomaticIn particular cases option of experimental therapyHLA-typing of the familyif no sibling donor available, consider unrelated donorClose follow-up of the patientThree monthly

89. Take home messageMore complete diagnosic workup is needed, when therapeutic intervention is an optionDistinction between different lymphoid neoplasmsDetermination of prognostic factorsNew biological prognostic markers do not directly determinate the time of treatmentThey may have an impact on the long term treatment strategyClinical staging still plays a essential role

90. Case 6History/ Clinicalfindings53 year old, female Presenting a transient ischemic attack (TIA) BrachiofacialhemiparesisRisk factors, dyslipidemia, and cigarette smokingWeight loss of 7kg with 5 monthsDiscrete splenomegaly

91. Initial blood counts

92. What is the most relevant change in peripheral blood?Leukocytosis Macrocytic anemia ThrombcytosisRegenerative anemiaPancytopenia

93. Peripheral blood

95. What do the changes in peripheral blood suggest?Status post-spenectomy Hemolytic anemia Marrow fibrosisMyeloproliferative disease Acute myeloid Leukemia

96. Bone marrow:dry tap

97. Bone marrow Histology