New Frontiers in Pathology Hematopathology Break-out Session

1. New Frontiers in PathologyHematopathology Break-out Session Bryan Coffing, M.D. Hematopathology Fellow

2. Disclaimer The cases reviewed here have been slightly modified for illustrative purposes Terms borrowed from the 2008 WHO blue book have been formatted to fit our lexicon (Leukaemia ? Leukemia, etc) I want to thank you all for being here. I wasn’t sure if we’d have an audience when I saw that we were presenting simultaneously with Dr. Appelman this afternoon.I want to thank you all for being here. I wasn’t sure if we’d have an audience when I saw that we were presenting simultaneously with Dr. Appelman this afternoon.

3. Goals Examine two cases of acute leukemia with ambiguous lineage Discuss the specificity of “lineage specific” markers in acute leukemia Compare the European Group for the Immunological Characterization of Leukemia (EGIL) schema to the new WHO guidelines for lineage designation Summarize a few of the characteristics of the acute leukemias of ambiguous lineage The World Health Organization recently came out with an updated version of their classification of tumors of hematopoietic and lymphoid tissues. You’ve no doubt noticed the increase in thickness over the previous version. It used to be that subspecialty hematopathologists joked about the complex classification being job security, but more and more I think it is the authors that are doing most of the laughing. Even dedicated hematopathologists have a hard time keeping up. The new book contains 367 pages of non-reference text, and you’re on page 30 before you’re even out of the explanation of rationale and summary of changes section. But this afternoon I’d like to take one section of the WHO classification and examine it a little more closely. Ironically, this section covers neoplasms that I find to be rather ambiguous: the acute leukemias of ambiguous lineage. The World Health Organization recently came out with an updated version of their classification of tumors of hematopoietic and lymphoid tissues. You’ve no doubt noticed the increase in thickness over the previous version. It used to be that subspecialty hematopathologists joked about the complex classification being job security, but more and more I think it is the authors that are doing most of the laughing. Even dedicated hematopathologists have a hard time keeping up. The new book contains 367 pages of non-reference text, and you’re on page 30 before you’re even out of the explanation of rationale and summary of changes section. But this afternoon I’d like to take one section of the WHO classification and examine it a little more closely. Ironically, this section covers neoplasms that I find to be rather ambiguous: the acute leukemias of ambiguous lineage.

4. Case Presentation #1 64 year old male with hypertension, diabetes, coronary artery disease Seen for routine follow-up in cardiology CBC: WBC 4.4; Hgb 8.1; Plt 76

5. Clinicians always want to know, are they myeloid or lymphoid? You probably have to get burned a couple of times before you stop guessing. I’ve been wrong once so far…once out of three guesses, so I still try to tell them.Clinicians always want to know, are they myeloid or lymphoid? You probably have to get burned a couple of times before you stop guessing. I’ve been wrong once so far…once out of three guesses, so I still try to tell them.

6. Additional history Patient reports: Fatigue for 2-3 months Persistent sinus infection for 4-6 weeks Easy bruising Referred to hematology for further workup Bone marrow biopsy, aspirate Flow cytometry Cytogenetic and molecular analysis

10. Ancillary Testing Molecular testing negative Cytogenetic analysis Complex abnormalities, multiple clones Hypotetraploid with del(5), del(21), add(13) Abnormality seen in AML as well as ALL

11. Flow Cytometry

12. Flow Cytometry

13. Flow Summary Positive CD34 (mod) CD33 (dim, sub) CD117 (dim-mod) CD38 (dim) CD45 (dim) cCD22 (dim, sub) cCD79a (dim, sub) Tdt (dim, sub) 5-7% MPO (+) by IHC Negative CD19 CD20 CD10 CD3 (surf & cyto) Other T cell markers Monocytic markers

14. Differential Diagnosis AML, without maturation (M1) B Lymphoblastic Leukemia/Lymphoma (with aberrant expression of MPO?) Acute leukemia of ambiguous lineage Biphenotypic/Bilineage (B-cell and myeloid)

15. Acute Leukemias of Ambiguous Lineage WHO 2001 3 categories (Undiff, Biphenotypic, Bilineage) Pages 106-107 WHO 2008 Combined two categories (Biphenotypic and Bilineage) Pages 149-155 Created 6 new categories

17. Lineage Assignment European Group for the Immunological Characterization of Leukemias (EGIL) Higher score given to those markers that are specific for lineageHigher score given to those markers that are specific for lineage

18. B Lymphoid Markers in AML CD7 and TdT expression in AML is well known CD79a expression in AML Cruse 2005: 4/46 (8.7%) Tiacci 2004: 10/160 (1.6%) El-Sissy 2006: 1/34 (2.9%) CD19 expression Tiacci 2004: 10/160 (1.6%) El-Sissy 2006 4/34 (11%) CD22 (EGIL score 2) El-Sissy 2006 1/34 (2.9%) CD7 in AML with monocytic differentiation, Tdt in AML M0CD7 in AML with monocytic differentiation, Tdt in AML M0

19. Lymphoid markers in AML t(8;21)(AML1;ETO) CD19 cCD79a Weak TdT More than 30% show PAX5 expression

20. AML with cytogenetic abnormalities AML with inv(16) or t(16;16) CD2 expression AML with t(15;17) CD2 expression in microgranular variant AML with t(6;9) 50% TdT expression M1 and M2 (AML, NOS) 10-20% positive for CD2, CD4, CD19, or CD56

21. Aberrant Markers in ALL Park et al, 1992 137 ALL cases in adults Phenotyped by immunofluoresence B-ALL more often CD13 (1%) and CD33 (2%) CD2 (1%) and CD5 (1%) T-ALL CD13 (8.3%) CD10 (16.7%)

22. Myeloid markers in ALL Kalina et al, 2005 381 B-ALL pediatric patients Flow cytometric immunophenotyping CD33 (23%) CD15 (20%) CD13 (16%) Vitale et al, 2007 374 adult ALL patients Flow cytometric immunophenotyping of CD13 and/or CD33 T-ALL (24%) B-ALL (38%)

23. Myeloid Markers in ALL MPO in pediatric ALL Austin 1998: (COG) 32/57 (56%) pediatric ALL expressed MPO at protein or mRNA level All cases negative for t(9;22) MPO in adult ALL Arber 2001: 19/67 (23%) positive 8/19 (42%) with bcr/abl Polyclonal MPO antibody Immunocytochemistry with monoclonal MPO negative

24. Most specific lineage markers Distinctly difficult to find lymphoblastic leukemias with aberrant MPO Burkitt like ALL Myeloid leukemia with aberrant CD3 also distinctly uncommon B cell markers seem to be most promiscuous

25. Myeloid lineage Flow cytometry Immunohistochemistry Cytochemistry Monocytic differentiation

26. T lineage Flow cytometry Immunohistochemistry Surface CD3

27. B lineage + At least ONE (strong): CD79a Cytoplasmic CD22 CD10 Weak CD19

28. MPAL - Exclusions recurrent AML-associated translocations - t(8;21) – freq expresses B cell markers - t(15;17) - inv (16) FGFR1 mutation associated leukemias CML blast crisis MDS-related AML therapy-related AML FGFR1 cell of origin is a pluripotent lymphoid-myeloid hematopoietic stem cell CML blasts often mixed phenotype: many MPO expressing ALLs found to harbor bcr/abl transcript and therefore likely CMLFGFR1 cell of origin is a pluripotent lymphoid-myeloid hematopoietic stem cell CML blasts often mixed phenotype: many MPO expressing ALLs found to harbor bcr/abl transcript and therefore likely CML

29. Case Presentation #2 47 year old male with cellulitis Transferred to University of Michigan for pancytopenia and blasts on blood smear CBC: WBC 2.9; Hb 8.6; Plt 71

32. Ancillary Studies Molecular Negative for bcr/abl Cytogenetics 11q deletion, del(18) FISH for MLL (11q23) rearrangements negative

33. Flow Cytometry

34. Flow Cytometry

35. Flow Summary Positive Cytoplasmic CD3 CD5 CD7 Tdt CD11c CD13 CD33 CD34 CD38 CD45 CD117 Cytoplasmic MPO Negative CD2 CD3 surface CD4 CD8 CD10 CD14 CD19 CD20 CD33 CD56

36. Differential Diagnosis AML with maturation (M2) T Lymphoblastic Leukemia/Lymphoma (with aberrant expression of myeloid markers) Acute leukemia of ambiguous lineage Biphenotypic/Bilineage (T-cell and myeloid)

37. References Arber, Daniel et al. Myeloperoxidase Immunoreactivity in Adult Acute Lymphoblastic Leukemia. Am J Clin Pathol 2001;116:25-33. Austin G et al. Prevalence of myeloperoxidase gene expression in infant acute lymphocytic leukemia. Am J Clin Pathol. 1998;110:575-581. Cruse, Julius et al. Aberrant expression of CD7, CD56, and CD79a antigens in acute myeloid leukemias. Experimental and Molecular Pathology 79 (2005) 39– 41. El-Sissy et al. Aberrant Lymphoid Antigen Expression in Acute Myeloid Leukemia in Saudi Arabia. Journal of the Egyptian Nat. Cancer Inst., Vol. 18, No. 3, September: 244-249, 2006. Gibson, et al. Expression of the B Cell–Associated Transcription Factors PAX5, OCT-2, and BOB.1 in Acute Myeloid Leukemia. Associations With B-Cell Antigen Expression and Myelomonocytic Maturation. Am J Clin Pathol 2006;126:916-924. Huh YO, McCulloch EA, Buhring H-J, et al: c-kit in leukemia and myelodysplasia as detected by monoclonal antibodies. Blood 80 (suppl):25a, 1992 Kalina, Thomas et al. Myeloid antigens in childhood lymphoblastic leukemia:clinical data point to regulation of CD66c distinct from other myeloid antigens. BMC Cancer. 5(38), 2005. Park et al. Acute Leukemias with Unusual Phenotypes. Journal of Korean Medical Science. 7(4), 377-384, 1992. Preti A, Huh YO, O'Brien SM, et al: Myeloid markers in adult acute lymphocytic leukemia. Cancer 76:1564, 1995. Tiacci et al. PAX5 Expression in Acute Leukemias: Higher B-Lineage Specificity Than CD79a and Selective Association with t(8;21)-Acute Myelogenous Leukemia. Cancer Research. 64, 7399–7404, October 15, 2004 Valbuena, Jose et al. Expression of B Cell–Specific Activator Protein/PAX5 in Acute Myeloid Leukemia With t(8;21)(q22;q22). Am J Clin Pathol 2006;126:235-240. Vitale, Antonella et al. Absence of prognostic impact of CD13 and/or CD33 antigen expression in adult acute lymphoblastic leukemia. Results of the GIMEMA ALL 0496 trial. Haematologica, Vol 92, Issue 3, 342-348.