1. ICCS e-Newsletter CSI Spring 2015 Prashanti Reddy, M.D., M.S. Senior Staff Hematopathologist Genoptix, Inc., a Novartis company

2. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI - Clinical History: 2 A 70-year-old male presented with a history of fever. Review of the peripheral blood smear at an external facility, had shown numerous blasts and nucleated red blood cells. No CBC data were available for review. Bone marrow was submitted for evaluation of acute leukemia.

3. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI - Flow Cytometric Analysis: 3 TubeFITCPEPerCP cy5.5PE cy7APCAPC cy7 1KappaCD38CD19CD5CD23CD45 2LambdaKappaCD19CD10CD20CD45 3FMC7CD22CD19CD5CD20CD45 4CD103CD257AADCD19CD11cCD45 5cMPOcCD79acCD3CD34TdTCD45

4. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI - Flow Cytometric Analysis: 4 A large population is detected in dim CD45 area with low side scatter properties

5. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI - Flow Cytometric Analysis: 5 This population is positive for CD19, CD10, and CD20 (dim), and shows dim to moderate CD38 expression

6. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI - Flow Cytometric Analysis: 6 This population is also positive for HLADR, CD22 (dim), and cytoplasmic CD79a

7. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI - Flow Cytometric Analysis: 7 This population shows no surface light chain expression and is negative for cytoplasmic CD3, TdT, and MPO

8. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI – Morphology: 8 Aspirate smear showing sheets of immature cells Core biopsy showing markedly hypercellular marrow

9. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 9 Precursor B-cell acute lymphoblastic leukemia/lymphoma Burkitt lymphoma (BL) with atypical immunophenotype High grade B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma (DLBCL) and BL e-CSI - Differential Diagnosis:

10. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI – Cytogenetics: 10

11. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. e-CSI – Cytogenetics: 11 t(14;18) t(8;14)

12. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 12 KARYOTYPE: 46,XY,t(3;22)(q27;q11.2),t(8;14)(q24;q32),t(14;18)(q32;q21)[16] /46,XY[4] NHL FISH: ABNORMAL results with t(8;14), t(14;18), MYC, and BCL6 rearrangements. e-CSI - Cytogenetic Results:

13. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 13 High-grade mature B-cell lymphoma best classified as B-cell lymphoma, unclassifiable with features intermediate between DLBCL and BL (triple hit lymphoma) e-CSI - Final Diagnosis:

14. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 14 B-cell lymphomas, unclassifiable with features intermediate between DLBCL and BL are aggressive lymphomas that have morphological and genetic features of both DLBCL and BL B-cell lymphomas with concurrent IGH-BCL2 and MYC translocations (double hit lymphomas) are rare and even less frequent are B-cell lymphomas with IGH-BCL2, MYC and BCL6 translocations (triple hit lymphoma) The relative incidence of double-hit and triple-hit lymphomas increases with age, up to more than 30% in elderly patients e-CSI - Intermediate BL/DLBCL:

15. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 15 Mainly diagnosed in adults Usually present with lymphadenopathy or widespread extranodal disease Can arise denovo or present with a prior history of follicular lymphoma Although rare, some patients present with a leukemic presentation e-CSI - Clinical Presentation of Double Hit and Triple Hit Lymphomas:

16. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 16 Present with poor prognostic parameters Advanced stage B symptoms Elevated LDH Poor performance status BM and CNS involvement High IPI (International Prognostic Index) score e-CSI - Clinical Presentation of Double Hit and Triple Hit Lymphomas:

17. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 17 CD19+ CD20+ (can be dim) CD22+ CD79a+ CD45+ (can be dim) CD10+ SIg+/- (SIg maybe negative in double hit and triple hit cases) TdT- BCL2+ CD34- High Ki-67 (>80%) e-CSI - Immunophenotype of Double Hit and Triple Hit Lymphomas:

18. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 18 All neoplasms usually show a complex karyotype with ≥3 numerical or structural aberrations Gene profiling studies show that some double hit lymphoma cases have a profile intermediate between BL and DLBCL or more similar to BL e-CSI – Cytogenetics:

19. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 19 Lymphomas with MYC/BCL2 protein co-expression (double- expressing lymphomas) are associated with a poor prognosis. However there is a significant discordance in the degree of immunohistochemical positivity with cut off values ranging from 40-50% for MYC and 30-70% for BCL2. Further clinical studies are warranted to establish a clear cut off value. e-CSI - Protein Expression:

20. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 20 These are high grade lymphomas with poor outcome, harboring double and triple chromosomal rearrangements, usually of MYC, BCL2, and BCL6. The combination of a potent proliferation signal (MYC) and a potent survival signal (BCL2) leads to a very aggressive clinical behavior and chemo-resistance. In addition, BCL6 rearrangements are also associated with a worse prognosis. e-CSI – Double Hit and Triple Hit Lymphomas:

21. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 21 Double hit and triple hit lymphomas with leukemic presentations mimicking B-acute lymphoblastic leukemia are rare. Diagnostic challenges arise when the initial presentation of the neoplastic B cells shows a phenotype with bright CD10, dim CD45, and lacking surface light chain expression, mimicking B lymphoblasts. Interpreting the immunophenotype in isolation may lead to a missed diagnosis. e-CSI – Conclusion:

22. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 22 There are no clinical, morphologic or immunophenotypic features to identify double hit and triple hit lymphoma cases. When Ki67 is >80%, FISH testing should be done for MYC rearrangement. In addition, determination of BCL2 and BCL6 rearrangement is helpful to identify double hit and triple hit lymphomas, as these are clinically aggressive and portend a poor prognosis. Double hit and triple hit lymphomas usually have a poor response to conventional chemotherapy and novel therapeutic approaches may have to be considered. e-CSI – Conclusion:

23. ©2014 Genoptix, Inc. All rights reserved. Genoptix ® is a registered trademark of Genoptix, Inc. GMAP14-013. 23 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (Eds.): WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues. IARC: Lyon 2008. Snuderl M, Kolman OK, Chen YB, Hsu JJ, Ackerman AM, Dal Cin P, Ferry JA, Harris NL, Hasserjian RP, Zukerberg LR, Abramson JS, Hochberg EP, Lee H, Lee AI, Toomey CE, Sohani AR. B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma and diffuse large B-cell lymphoma. Am J Surg Pathol. 2010;34(3):327–340. Wu D, Wood BL, Dorer R, Fromm JR. “Double-Hit” mature B-cell lymphomas show a common immunophenotype by flow cytometry that includes decreased CD20 expression. Am J Clin Pathol. 2010;134:258–265. Li S, Lin P, Fayad LE, Lennon PA, Miranda RN, Yin CC, Lin E, Medeiros LJ. B-cell lymphomas with MYC/8q24 rearrangements and IGH@BCL2/t(14;18)(q32;q21): an aggressive disease with heterogeneous histology, germinal center B-cell immunophenotype and poor outcome. Mod Pathol. 2012;2:145–156. Petrich AM, Nabhan C, Smith SM. MYC-associated and double-hit lymphomas: A review of pathobiology, prognosis, and therapeutic approaches. Cancer. 2014 Jul 24. e-CSI – References: