5003 Immunohistochemistry in hematopathology, what's in, what's out, what's useful

Transcription

1 Immunohistochemistry in hematopathology, what's in, what's out, what's useful Kathryn Rizzo, DO, PhD VIRGINIA COMMONWEALTH UNIVERSITY Department of Pathology School of Medicine Mehdi Nassiri, MD INDIANA UNIVERSITY Department of Pathology and Laboratory Medicine School of Medicine

2 Speaker Disclosure In the past 12 months, we have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in our presentation.

3 Course Objectives To become familiar with new IHC markers. To choose the correct antibodies based on the morphologic, clinical and laboratory findings. Create an integrated comprehensive report with correct terminology, and communicate the findings effectively to clinical team.

4 Diagnostic Approach Pattern or Panel

5 Architecture Diffuse Nodular Small Large Mixed Small Large Mixed

6 Architecture Diffuse Nodular B T Mixed B T Mixed

7 Useful Morphologic Findings Diagnosis Low Power Nuclei CLL/SLL MZL Diffuse/Vaguely nodular Proliferation center Marginal zone Interfollicular Monocytoid cells Follicular colonization Round Irregular MCL Diffuse/Vaguely nodular Mantle zone Irregular FL Monotonous Follicles Cleaved

8 SLL MZL MCL FL

9 Practical Immunophenotypic Panel (in addition to CD3, CD20, Kappa, Lambda/clonality) Diagnosis CD5 CD10/ BCL6 SLL CD23 Cyclin D MZL MCL FL

10 Practical Immunophenotypic Panel New Markers Diagnosis LEF1 SOX11 SLL MZL MCL FL * - - * Negative in indolent MCL

11 Is CD20 always useful Can be weak- CLL/SLL Can be negative if treated with Rituxan Can be positive- MM with t(11;14)

12 Other B cell markers CD79a- is positive in plasma cells PAX5- nuclear stain, can be variable Can be positive in small cell ca/neuroendocrine ca CD19 CD22

13 SOX11 SRY (sex determining region Y)-box 11 Transcription factor % CCND1-pos MCL 100% CCND1-neg MCL Can be positive in B-LBL,TLBL, Burkitt s, T-PLL-depends on the Ab! Negative in DLBCL

14 IHC in Differential Diagnosis of HCL SMZL SRPL HCL-V HCL Cyclin D1 neg neg neg pos CD123 neg neg neg pos CD25 var var neg pos Annexin neg neg neg pos TRAP neg neg var pos BRAF neg neg neg pos

15 BRAFV600E Melanoma Hairy cell leukemia

16 Bcl-2 Immunohistochemistry Follicular Hyperplasia Follicular Lymphoma

17 Lineage CD3, PAX5/CD19, MPO Stem cells/precursors Bone Marrow IHC CD34, CD117, CD123,TdT, CD1a Myeloid MPO, Lyzozyme, CD13, CD33 Monocytic CD14, CD68 (KP-1,PGM-1), CD163 Erythroid Glycophorin, hemoglobin, E-cadherin Megakaryocytic CD42b, CD61, von Willebrand s factor (factor VIIIRA)

18 Bone Marrow IHC Acute leukemias CD34, CD117, TdT, CD123, CD1a CD3, CD19, PAX5 MPO, lysozyme, CD10, CD13, CD33, CD61, CD42b, hemoglobin A, glycophorin, CD68, CD14

19 Bone Marrow IHC Myelodysplastic syndromes CD34, CD117 TP53 Mastocytosis Mast cell tryptase, CD117, CD25, CD2

20 Predictive Markers CD20-RITUXIMAB CD30-Brentuximab vedotin relapsed CHL and relapsed salcl MYC, KI-67, etc

21 Immunohistochemistry of normal and abnormal plasma cells. Normal Abnormal PAX5 Neg. Pos. (CYCLIND1) CD20 Neg. Pos. (subset) CD13 Neg. Pos. (subset) CD33 Neg. Pos. (subset) CD56 Neg. Pos. (most) CD117 Neg. Pos. (subset) CYCLIND1 Neg. Pos. (subset) P53 Neg. Pos. (subset)

22 IHC analysis of Diffuse Large B cell Lymphoma Determine Cell Of Origin Germinal center B cell type versus Activated B cell type IHC algorithms - Hans - Choi - Tally

23 DLBCL Hans CD10+ GCB CD10- BCL6- ABC CD10- BCL6+ MUM1- GCB CD10- BCL6+ MUM1+ ABC GCB = germinal center B cell type ABC = activated B cell type

24 DLBCL cell of origin subtypes Why is it important? Prognosis 5 yr survival GCB Good >50% ABC Bad 30% PMBCL Good >60% Treatment: R-CHOP vs R-EPOCH vs NF-KB targeted therapy

25 Immunophenotypic features in DLBCL: GCB, ABC, and primary mediastinal DLBCL (PMBL) GCB-DLBCL ABC-DLBCL PMBL CD10 BCL6 LMO2 SERPIN A9 (GCET1) HGAL(GCET2) MUM1/IRF4 BCL2 FOXP1 CCNE CCND2 SCYA2 MALT1 XBP1 REL MAL CD30 MUM1 FIG1 TRAF1

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28 DLBCL vs PMBCL DLBCL PMBCL CD23 ~10% ~70% TRAF ~10% ~60% CREL ~20% ~60% CD200 ~90% ~10%

29 REL TRAF

30 Future of MYC in High grade B cell Lymphomas Flourescent in situ hybridization: myc, bcl-2, bcl-6 Immunohistochemistry: myc and bcl-2 overexpressers. Cut off for positive staining: Myc 40% Bcl2 30%

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32 Importance in myc/bcl2 overexpressors Higher proportion in ABC-DLBCL and in myc translocated tumors Also present in non-myc/bcl-2 translocated tumors Portends to a poor prognosis Poor concordance, seen in other tumors, difficult antibody

33 Double, Triple hit lymphomas Translocation of myc with bcl-2 and/or bcl-6 Portend to poor prognosis. Myc alone translocation portends to poor prognosis. No specific immunophenotype, more common in GCB, not correlated with ki-67 expression level. Need to differentiate Different treatment then DLBCL and BL.

34 Green T. JCO. 2012;30(28): Johnson N. JCO. 2012;30(28): Aukema S., BLOOD. 2011;117(8): Perry A. BJH doi:

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36 CD20 KI-67 BCL-2

37 FISH Positive for an MYC/IGH fusion representing a t(8;14) Negative for IGH/BCL6 Fusion Negative for IGH/BCL2 Fusion

38 MYC-complex karyotype Rare Common Rare overall BL BCLU DLBCL Ki67 > 90% and homogeneous Yes Common Uncommon Ki67 < 90% and heterogeneous Rare Sometimes Common BCL2 negative/weak Yes Sometimes Sometimes BCL2 strong No Sometimes Sometimes MYC rearrangement Yes Common Rare IGH-MYC Common Uncommon Uncommon Non-IGH-MYC Uncommon Common Common BCL2 pos MYC neg No Uncommon Sometimes BCL6 pos MYC neg No Uncommon Sometimes MYC-simple karyotype Yes Rare Rare

39 Differential Diagnosis CD45 CD15 CD30 CD20 PAX-5 Oct.2 BOB.1 PU.1 BCL-6 EBV NLPHL + - -/ * Classical HL /+ +wk +/- -/+ - -* +/- TC/HRLBCL +/- - -/ /+ - ALCL +/

40 IHC Hodgkin Lymphoma Ideal Hodgkin CD30+, CD15+, CD20- Reality: CD15-, CD20+ CD2 Additional useful markers PAX5 (weak), MUM1, CD45 T cell marker expression CD4

41 CD30 CD20 CD20 CD20

42 SUMMARY DLBCL should be investigated for the cell of origin based on IHC algorithm Differentiation of DLBCL versus BCLU (double-triple hit lymphomas) versus BL versus PMLBCL. IHC panel FISH myc, bcl-2, bcl-6

43 SUMMARY Classical Hodgkin lymphomas may have unusual immunophenotype. Focal CD20 expression, lack of CD15 NLPHL and Tcell/Histiocyte rich large B cell lymphoma. May have focal/weak CD30 expression Strong CD20 expression

44 Guideline From the College of American Pathologists Pathology and Laboratory Quality Center Fitzgibbons PL, et al Arch Pathol Lab Med. doi: /arpa CP 1- Laboratories must validate all IHC tests before placing into clinical service. Correlating the new test s results with the morphology and expected results; Comparing the new test s results with the results of prior testing of the same tissues with a validated assay in the same laboratory; Comparing the new test s results with the results of testing the same tissue validation set in another laboratory using a validated assay; Comparing the new test s results with previously validated non-immunohistochemical tests; or Testing previously graded tissue challenges from a formal proficiency testing program (if available) and comparing the results with the graded responses. 2- For initial validation of every assay used clinically, with the exception of HER2/neu, ER, and PgR (for which established validation guidelines already exist), laboratories should achieve at least 90% overall concordance between the new test and the comparator test or expected results. If concordance is less than 90%, laboratories need to investigate the cause of low concordance.

45 Guideline From the College of American Pathologists Pathology and Laboratory Quality Center Fitzgibbons PL, et al Arch Pathol Lab Med. doi: /arpa CP 3- For initial analytic validation of nonpredictive factor assays, laboratories should test a minimum of 10 positive and 10 negative tissues. When the laboratory medical director determines that fewer than 20 validation cases are sufficient for a specific marker (eg, rare antigen), the rationale for that decision needs to be documented. Note: The validation set should include high and low expressors for positive cases when appropriate and should span the expected range of clinical results (expression levels) for markers that are reported quantitatively. 4- For initial analytic validation of all laboratory-developed predictive marker assays (with the exception of HER2/neu, ER, and PgR), laboratories should test a minimum of 20 positive and 20 negative tissues. When the laboratory medical director determines that fewer than 40 validation tissues are sufficient for a specific marker, the rationale for that decision needs to be documented. Positive cases in the validation set should span the expected range of clinical results (expression levels). This recommendation does not apply to any marker for which a separate validation guideline already exists 5- For a marker with both predictive and nonpredictive applications, laboratories should validate it as a predictive marker.

46 Guideline From the College of American Pathologists Pathology and Laboratory Quality Center Fitzgibbons PL, et al Arch Pathol Lab Med. doi: /arpa CP 8- If IHC is regularly done on decalcified tissues, laboratories should test a sufficient number of such tissues to ensure that assays consistently achieve expected results. 9- Laboratories may use whole sections, TMAs, and/or MTBs in their validation sets as appropriate. Whole sections should be used if TMAs/MTBs are not appropriate for the targeted antigen or if the laboratory medical director cannot confirm that the fixation and processing of TMAs/ MTBs is similar to clinical specimens. 10- When a new reagent lot is placed into clinical service for an existing validated assay, laboratories should confirm the assay s performance with at least 1 known positive case and 1 known negative case.

47 Guideline From the College of American Pathologists Pathology and Laboratory Quality Center Fitzgibbons PL, et al Arch Pathol Lab Med. doi: /arpa CP 11- Laboratories should confirm assay performance with at least 2 known positive and 2 known negative cases when an existing validated assay has changed in any one of the following ways: Antibody dilution; Antibody vendor (same clone); Incubation or retrieval times (same method) Laboratories should confirm assay performance by testing a sufficient number of cases to ensure that assays consistently achieve expected results when any of the following have changed: Fixative type; Antigen retrieval method (eg, change in ph, different buffer, different heat platform); Antigen detection system; Tissue processing or testing equipment; Environmental conditions of testing (eg, laboratory relocation); Laboratory water supply. 13- Laboratories should run a full revalidation (equivalent to initial analytic validation) when the antibody clone is changed for an existing validated assay

48 REFERENCES Mozos A, et al. Haematologica 2009; 94: Fernandez V, et al. Cancer Res 2010;70(4); Ondrejka SL, et al. Haematologica 2011;96(8): Chen, Y-H, et al. Modern Pathology 2010;23: Zeng W, et al. Am J Surg Pathol. 2012;36(2):214-9 Soldini D, et al. Am J Surg Pathol. 2014;38(1):86-93 Tandon B, et al. Mod Pathol. 2011;24(11): Read JA, et al. Clin Lymphoma Myeloma Leuk pii: S (14) Gualco G, et al. Mod Pathol. 2012;25(11): Ballabio E, et al. Clin Cancer Res. 2010;16(14): Choi WW, et al. Clin Cancer Res. 2009;15(17): Hans CP, et al. Blood. 2004;103(1): Muris JJ, et al. J Pathol. 2006;208(5): Nyman H, et al. Mod Pathol. 2009;22(8): Meyer PN, et al. J Clin Oncol. 2011;29(2):200-7 Aukema S, et al. BLood. 2011;117(8): Perry A, et al. BJH doi: Green T, et al. JCO. 2012;30(28): Johnson N, et al. JCO. 2012;30(28): Cook J, et al. AJSP. 2014;38: Hill B, et al. Leukemia lymphoma. 2012;53(5): Alizadeh A, et al.nature.2000:403: Rosenwald A, et al. Blood. 2000;95: Jost P, et al. Blood. 2007;109: Browne P, et al. AJCP. 2003;120: Garcia-Cosio M, et al. Mod Path. 2004;17: Carbone A, et al. BJH. 2002;177: McCune R, et al. Mod Path. 2006;19: Jundt F, et al. Blood.2002;99: Hu S et al. Blood. 2013;121: Li S, et al. Mod Path. 2012;25: