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

2. Case 1 A 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. Scattergram PEROX: 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 smear smallblastswithscantcytoplasm, condensednuclearchromatinandindistinctnucleoli, andlarger blastswithmoderate cytoplasm, smallcytoplasmicvacuolesand a distinctnucleoli. Noprimarygranulesor Auer rods 35% blasts = acuteleukemia

6. BM Cytology no 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 Histology Giemsa 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 Cytometry Blasts: 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 EGIL mature B Cell neoplasms Mature B Common B Pre-B pro B AUL TdT CD19 CD10 cIgM sIgM IgG/IgD/ Kappa/Lambda

10. MolecularGenetics Multiplex 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 prephase Chemosensitivityevaluation d+8 Double induction R – Rituximabif CD20+ R – HyperCVADvsconventional Monitor MRD / B-cellreceptorgenerearrangement / flow / fusiontranscripts

12. Treatment on GRAALL Transplant in CR1 ? WBC > 30G/L CNS involved B-ALL: CD10-; t(4;11); t(1;19) Complexcaryotype (>5); haploid ornear triploid Corticoresistance Chemoresistance No CR1 after induction

13. Case 2 A 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. Scattergram Perox: 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 Blood small 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. MarrowCytology increased 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. Histo cellularity 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. Flow Blasts, 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. Diagnosis Molecular 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 3 A 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. MarrowCytology The 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. Histology marrow 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 monthslater No complaints, no treatment proposed 6 months later: fatigue, no fever, no bleeding.

28. Hem Lab follow-up

29. Blood Smear @ FU Evident 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. MarrowCytology Marrow 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. Histology Densely 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. Genetics Only 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 dysplasia with ringed sideroblasts and cytogenetic anomalies

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

34. Cytogenetics 12 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 related with 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 4 50-year old female patient Progressive fatigue since 4 months Diffuse upper abdominal pain No history of radiotherapy / drug exposure Slight splenomegaly blood count

37. Blood count

38. Peripheral blood smear

39. At this step what is the most probable diagnosis ? SeverebacterialInfection Myeloproliferativedisorder (MPD) Chronicmyeloidleukemia (CML) MPD / MDS None oftheabove

40. What is typical for CML? Leukocytosis Neutrophilia with pathologic left shift Increased number of myelocytes/metamyelocytes Thrombocytosis Anisocytosis of platelets Increased number of eosinophils Increased number of basophils

41. What is absolutely necessary to confirm the diagnosis Flowcytometry Bone marrow histology including immunohistchemistry Cytogenetics Molecular genetics Bone marrow cytology

42. Molecular genetic result Bcr/abltranscript positive b2a2 Do we still needconventionalcytogenetics?

44. Why do we need morphology in CML patients ? Confirmation of the diagnosis of CML Define the stage of the disease To complete the diagnosis To recognize possible atypical forms of CML or MPD For treatment decision

45. Bone marrow cytology

46. Bone marrow histology Hematoxylin-eosin stain Gömörri stain (silver stain)

47. Immunohistochemistry (anti CD34) 40x 20x

48. Stages of the CML Atdiagnosis Chronicphase Acceleratedphase Blast crisis Myeloid blast crisis Lymphoid blast crisis Duringfollow-up Remission Hematological Morphological Cytogenetic Major Molecular CompleteMolecular Chronicphase Acceleratedphase Blast crisis

49. Additional information Bonemarrowcytology Blasts 1.5%, Basophils1% Bonemarrowhistology CD34 positive cells 2.5% Cytogenetics 46,XX,t(9;22)(q34;q11)[20]

50. What is the stage of the disease at diagnosis? Myeloid blast crisis Hematological remission Chronic phase Accelerated phase None of the above

52. Peripheralbloodbasophils> 20%

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

54. orthrombocytosis (>1000x109/l) unresponsivetotherapy

55. Increasingspeensizeandincreasing WBC unresponsivetotherapy

57. Final diagnosis Chronic myeloid leukemia (CML) t(9;22)(q43;q11)/BCR-ABL positive in chronic phase

58. How would you treat this patient?

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

63. Peripheral blood smear

64. Diverse mature lymphoid neoplasms B-CLL Hairy cell leukemia Sezary‘s Syndrome Follicular lymphoma T-LGL leukemia Prolymphocytic leukemia

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

68. Immunoglobulin levels normal

69. no monoclonal fraction

71. Immunophenotyping results

72. Algorithm of mature B-cell neoplasms Monoclonal B-cells + - CD5 + + - - CD23 CD10 others CLL MCL FL

73. Complete result of the immunophenotyping Kappa-monoclonality Cells are positive for CD19+, CD20+, CD5+, CD23+ Negative or dim positive for sIgM dim, CD79b-, FMC7-, CD10-, CD103- Immunophenotypic conclusion Mature B-cell neoplasm, Type B-CLL CLL score (matutes score) 5/5

74. Final diagnosis Mature B-cell lymphoid neoplasia, type B-CLL Rai 0, Binet A asymptomatic No autoimmune phenomena In an elderly patient (79-years old) No genetic information available

75. What will be the treatment strategy? Watch and wait Regular follow-up (every 6 months) Constitutional symptoms Organomegaly Peripheral blood count Why is it acceptable to restrict diagnostics to a minimal program? elderly patient no intention of curative therapy asymptomatic patient no treatment required

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-CLL Complete Immunophenotype Lymphocytes positive for CD19, CD20dim, CD23, CD5, kappa, sIgM dim Lymphocytes negative for: FMC-7, CD79b, CD103, CD10

82. How to define management strategy Young patient with B-CLL Rai 0, Binet A Considerations for management strategy Age of the patient Age - not an independent risk factor in CLL Younger patients are more likely to die from CLL Stage of the disease: early stage is due to early stage with undefined prognosis or good prognosis CLL – smoldering disease We need more information

83. Clinical prognostic factors in CLL Symptomatic CLL; B-symptoms Fever, night sweat, weight loss Disease stage Rai or Binet Lymphocyte count > 50 x109/L Doubling time of absolute lymphocyte count < 12 months

84. Genetics -> Prognosis interphase FISH Good prognosis Isolated del 13q14 Intermediate Trisomy 12 Normal karyotype Poor prognosis del 11q del17p del 11q

85. Mutational status of IgVH genes in B-cell CLL Two phases in B-cell development Antigen-independent phase Bone marrow Gene rearrangement Lymphoid follicle (follicular mantle) Unmutated B-cells naïve B-cells Antigen-dependent phase In lymphoid follicle (germinal centre) Somatic hypermutation mutated B-cells Memory cells

86. Biological surrogate markers for mutational status Mutational status not routinely available CD38 expression on B-cells Poor prognosis: positive ( 30%) No good correlation with mutational status ZAP70 expression on B-cells Poor prognosis: positive (>20%) Better correlation

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

88. Interpretation of the global situation and next steps Early stage, high risk B-CLL in a young patient CLL asymptomatic Rai I / Binet A stage, lymphocyte count FISH del(11q); ZAP-70 positive, CD38 positive Watch and wait as long as asymptomatic In particular cases option of experimental therapy HLA-typing of the family if no sibling donor available, consider unrelated donor Close follow-up of the patient Three monthly

89. Take home message More complete diagnosic workup is needed, when therapeutic intervention is an option Distinction between different lymphoid neoplasms Determination of prognostic factors New biological prognostic markers do not directly determinate the time of treatment They may have an impact on the long term treatment strategy Clinical staging still plays a essential role

90. Case 6History/ Clinicalfindings 53 year old, female Presenting a transient ischemic attack (TIA) Brachiofacialhemiparesis Risk factors, dyslipidemia, and cigarette smoking Weight loss of 7kg with 5 months Discrete splenomegaly

91. Initial blood counts

92. What is the most relevant change in peripheral blood? Leukocytosis Macrocytic anemia Thrombcytosis Regenerative anemia Pancytopenia

93. Peripheral blood

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

96. Bone marrow:dry tap

97. Bone marrow Histology

103. Summary of morphology Aspiration: Punctio sicca Hypercellular, maturating marrow without increased blasts Clustered Megakaryocytes with variable size Reticulin fibers moderately augmented Dilated sinusoids with prominent intracellular megakaryocytes Stainable iron Lymphoid follicles

104. What is the diagnosis at this stage? Essential thrombocythemia (ET) Chronic myeloproliferative disease (MPD) Chronic idiopathic myelofibrosis (CIMF) Chronic myeloproliferative disease and suspicion for Non-Hodgkin-Lymphoma CML No diagnosis possible, further staining required

105. Do we need additional investigations? Immunocytochemistry: CD34 staining Cytogenetics and BCR-ABL fusion transcript Colony forming assay JAK2 mutational status All of them

106. Results of additional investigations Immunohistochemistry no CD34-positive cells, no increase of blasts Genetic analysis 46,XX[20] BCR-ABL negative JAK2-V617F mutated Colony forming assay No erythroid independent BFU’s

109. Definitive diagnosis Chronicidiopathicmyelofibrosis (CIMF) in earlyfibroticstage no blast increase reactivelymphocyticinfiltrate Goodprognosticfeatures fewconstitutionalsymptoms nocytopenia (except a mild anemia) no blast excess nocytogeneticanomalies Thromboemboliccomplications (TIA)

110. Treatment?

111. ThankYou