www.ascp.org/ascp2014

5003 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

www.ascp.org/ascp2014

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.

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.

Diagnostic Approach

Pattern or

Panel

Architecture

Diffuse Nodular

Small Large Mixed Small Large Mixed

Architecture

Diffuse Nodular

B T Mixed B T Mixed

Useful Morphologic Findings

Diagnosis Low Power Nuclei

CLL/SLL Diffuse/Vaguely nodular Proliferation center

Round

MZL Marginal zone Interfollicular Monocytoid cells

Follicular colonization

Irregular

MCL Diffuse/Vaguely nodular Mantle zone

Irregular

FL Monotonous Follicles Cleaved

SLL MZL

FL MCL

Practical Immunophenotypic Panel (in addition to CD3, CD20, Kappa, Lambda/clonality)

Diagnosis CD5 CD10/ BCL6

CD23 Cyclin D1

SLL + - + - MZL - - - - MCL + - - + FL - + - -

Practical Immunophenotypic Panel New Markers

Diagnosis LEF1 SOX11

SLL + - MZL - - MCL - +* FL - -

* Negative in indolent MCL

Is CD20 always useful

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

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

SOX11 SRY (sex determining region Y)-box 11

Transcription factor

• 70-100% 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

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

BRAFV600E

Melanoma Hairy cell leukemia

Bcl-2 Immunohistochemistry

Follicular Hyperplasia Follicular Lymphoma

Bone Marrow IHC • Lineage

– CD3, PAX5/CD19, MPO

• Stem cells/precursors – 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)

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

Bone Marrow IHC

• Myelodysplastic syndromes – CD34, CD117 – TP53

• Mastocytosis

– Mast cell tryptase, CD117, CD25, CD2

Predictive Markers

• CD20-RITUXIMAB • CD30-Brentuximab vedotin

– relapsed CHL and relapsed sALCL

• MYC, KI-67, etc

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)

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

DLBCL Hans

CD10+ GCB

CD10- BCL6-

CD10- BCL6+

ABC

MUM1-

CD10- BCL6+ MUM1+

GCB

ABC

GCB = germinal center B cell type ABC = activated B cell type

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

GCB-DLBCL ABC-DLBCL PMBL

Immunophenotypic features in DLBCL: GCB, ABC, and primary mediastinal 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

DLBCL vs PMBCL

DLBCL PMBCL

• CD23 ~10% ~70% • TRAF ~10% ~60% • CREL ~20% ~60% • CD200 ~90% ~10%

TRAF REL

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%

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

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.

Aukema S., BLOOD. 2011;117(8):2319-2331

Johnson N. JCO. 2012;30(28):3452-3459 Green T. JCO. 2012;30(28):3460-3467

Perry A. BJH. 2014. doi:10.1111

CD20 KI-67 BCL-2

FISH

• Positive for an MYC/IGH fusion representing a t(8;14)

• Negative for IGH/BCL6 Fusion • Negative for IGH/BCL2 Fusion

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

MYC-complex karyotype Rare Common Rare overall

Differential Diagnosis

CD45

CD15

CD30

CD20

PAX-5

Oct.2

BOB.1

PU.1

BCL-6

EBV

NLPHL + -

-/+ + + + + + + -*

Classical HL - + +

-/+ +wk

+/- -/+

- -*

+/-

TC/HRLBCL +/-

- -/+ + + + + +

-/+ -

ALCL +/-

- + - - - - - - -

IHC Hodgkin Lymphoma

• Ideal Hodgkin – CD30+, CD15+, CD20-

• Reality: – CD15-, CD20+

• Additional useful markers − PAX5 (weak), MUM1, CD45

• “T cell marker” expression

CD2

CD4

CD30 CD20

CD20 CD20

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

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

Guideline From the College of American Pathologists Pathology and Laboratory Quality Center

Fitzgibbons PL, et al Arch Pathol Lab Med. doi: 10.5858/arpa.2013-0610-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.

Guideline From the College of American Pathologists Pathology and Laboratory Quality Center

Fitzgibbons PL, et al Arch Pathol Lab Med. doi: 10.5858/arpa.2013-0610-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.

Guideline From the College of American Pathologists Pathology and Laboratory Quality Center

Fitzgibbons PL, et al Arch Pathol Lab Med. doi: 10.5858/arpa.2013-0610-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.

Guideline From the College of American Pathologists Pathology and Laboratory Quality Center

Fitzgibbons PL, et al Arch Pathol Lab Med. doi: 10.5858/arpa.2013-0610-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). 12-. 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

REFERENCES Mozos A, et al. Haematologica 2009; 94:1555-1562 Fernandez V, et al. Cancer Res 2010;70(4); 1408–18. Ondrejka SL, et al. Haematologica 2011;96(8):1121-7 Chen, Y-H, et al. Modern Pathology 2010;23: 105–112 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):1433-43. Read JA, et al. Clin Lymphoma Myeloma Leuk. 2014. pii: S2152-2650(14)00140-2. Gualco G, et al. Mod Pathol. 2012;25(11):1439-45. Ballabio E, et al. Clin Cancer Res. 2010;16(14):3805-6. Choi WW, et al. Clin Cancer Res. 2009;15(17):5494-502. Hans CP, et al. Blood. 2004;103(1):275-82. Muris JJ, et al. J Pathol. 2006;208(5):714-23. Nyman H, et al. Mod Pathol. 2009;22(8):1094-101. Meyer PN, et al. J Clin Oncol. 2011;29(2):200-7 Aukema S, et al. BLood. 2011;117(8):2319-2331 Perry A, et al. BJH. 2014. doi:10.1111 Green T, et al. JCO. 2012;30(28):3460-3467 Johnson N, et al. JCO. 2012;30(28):3452-3459 Cook J, et al. AJSP. 2014;38:494-501 Hill B, et al. Leukemia lymphoma. 2012;53(5):763-769

Alizadeh A, et al.Nature.2000:403:503-511 Rosenwald A, et al. Blood. 2000;95:1797-1803 Jost P, et al. Blood. 2007;109:2700-2707 Browne P, et al. AJCP. 2003;120:767-777 Garcia-Cosio M, et al. Mod Path. 2004;17:1531-1538 Carbone A, et al. BJH. 2002;177:366-372 McCune R, et al. Mod Path. 2006;19:1010-1018 Jundt F, et al. Blood.2002;99:3060-3062 Hu S et al. Blood. 2013;121:4021-4031 Li S, et al. Mod Path. 2012;25:145-156