DARATUMUMAB HARD TO PRONOUNCE - EVEN HARDER ON BLOOD BANK TESTING. Dr. Jennifer Duncan Hematopathology Resident March 18, 2017

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1 DARATUMUMAB HARD TO PRONOUNCE - EVEN HARDER ON BLOOD BANK TESTING Dr. Jennifer Duncan Hematopathology Resident March 18, 2017

2 Goals and Objectives: Briefly describe the use of dara in the treatment of multiple myeloma Describe dara's interference in pretransfusion testing Discuss some ways to overcome this interference Review AABB recommendations Discuss UAH approach to these cases

3 WHAT IS MULTIPLE MYELOMA? Most common plasma cell malignancy 90% of cases occur in those > 50 years of age Plasma cells found primarily in bone marrow Role is to produce immunoglobulins Wide spectrum of disease Diagnosis is based on combination of lab, radiology and pathology finding Myeloma.org

4 SPEP MYELOMA CRAB Malignant plasma cells produce high amount of one type of immunoglobulin (M protein) Myeloma cells crowd out normal cells in marrow causing anemia Activate cells that destroy bone High calcium levels result from bone destruction High calcium and immunoglobulin damage kidneys over time Myeloma.org

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6 Not curable with current therapies, only treatable Newer treatment options have increased survival Typically triple drug regimens May include stem cell transplant depending on age and suitability Relapse and refractory disease is common AHS Clinical Practice Guidelines Multiple Myeloma 2015

7 DARA NEW KID ON THE BLOCK New anti-cd38 monoclonal antibody targeting myeloma cells Marketed as Darzalex by Janssen Pharmaceuticals FDA approval 2015, Health Canada approval 2016 Approved as third line therapy for multiple myeloma Given every four weeks until disease progresses

8 TARGET = CD38 CD38 is a membrane protein with multiple functions (enzyme, adhesion, signaling) Highly expressed on malignant plasma cells Also on lymphocytes, myeloid cells, red cells and other tissues

9 DARA - NOT JUST FOR MULTIPLE MYELOMA? Currently in trials to treat patients with earlier stage myeloma Early clinical trials involving non- Hodgkin's lymphoma Possible future role in AML?

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11 DARA A NEW PROBLEM IN BLOOD BANK CD38 is weakly expressed on red cells Daratumumab in patient plasma binds to CD38 on reagent red cells during testing Result is pan-reactivity with blood bank testing Problem identified during early trials with daratumumab Currently daratumumab is the only drug that has this effect Daratumumab more likely to be used in advanced disease more likely for these patients to need transfusion Chapuy et al, 2015 Transfusion 55:1545

12 Anti-CD38 DARATUMUMAB INTERFERENCE AHG Anti-CD38 CD38 AHG CD38 Zarandona M et al, CMAJ :3 Testing with IAT method will have pan-reactivity Agglutination can occur in all media and methods (saline, LISS, PEG, solid phase)

13 WHICH BLOOD BANK TESTS ARE AFFECTED? Plasma samples from treated patients can cause positive reactions in: Antibody Screens Antibody Identification Panels Serological Crossmatches +/- DAT (IgG), Eluate usually non reactive Dara does not interfere with : ABO, RhD typing Other antigen typing Immediate spin crossmatches

14 DOES THIS OCCUR WITH OTHER CD38 ANTIBODIES? Three other anti-cd38 antibodies were produced in trial IAT was performed with plasma spiked with these antibodies Found comparable dose-dependent agglutination patterns Conclusion: interference is not unique to dara but is classspecific problem for anti-cd38 Oostendorp et al, 2015 Transfusion 55: 1555

15 DOES DARA CAUSE IN VIVO HEMOLYSIS? DAT s may be negative or weakly positive Eluates prepared from patient s red cells do not agglutinate with other red cells or patients own red cells Suggest an in vivo clearance of small red cell fraction to which dara is bound Supported by a decrease in Hb (10 g/l) and increase in retic count found (although determined to be clinically insignificant) Oostendorp et al, 2015 Transfusion 55:1555

16 TYPICAL TESTING RESULTS WITH DARA ABO/Rh typing : no interference Antibody screen : all screening cells reactive Antibody panels: all panel cells reactive, auto control often negative DAT: variable AHG crossmatches: all rbc units tested will be incompatible Adsorptions: cannot eliminate pan reactivity **Patient s plasma can remain pan-reactive for 2-6 months after last daratumumab infusion

17 PROBLEM #1 - DARA IMPERSONATES AN ANTIBODY TO A HIGH INCIDENCE ANTIGEN High incidence antibodies are easy to find but difficult to identify Reactions are usually weak (1+) but stronger reactions (up to 4+) can be seen in solid phase testing. Not enhanced/destroyed by enzymes or affected by other enhancement media (LISS, PEG)

18 INVESTIGATING HIGH INCIDENCE ANTIGENS Specimens often referred to reference laboratory for further investigation Requires testing of patient s plasma with rare red cells Complex, time consuming investigations May cause further delay and higher costs Could be mistaken for clinically significant antibody to a Lutheran antigen as Lutheran null Lu(a-b-) cells appear to lack CD38. 1,2 1.Velliquette et al, 2015 Transfusion 2015: 55(3S) 26A 2. Aye et al, 2015 Transfusion 2015:55(3S):28A

19 PROBLEM #2 - DARA CAN MASK THE PRESENCE OF A SIGNIFICANT ALLO ANTIBODY Interference cannot be removed with adsorption All serological crossmatches are incompatible due to dara interference Further investigations to assist in ruling out alloantibodies may cause delays and use blood bank resources

20 DOES THIS OCCUR WITH ALL DARA PATIENTS? CBS - 6 patients in early 2014 started on dara as part of trial Pretreatment antibody screens negative in all at CBS (solid phase) 2 O positive, 2 A positive, 1 A negative, 1 B negative Results: 2 patients remained negative while on therapy 3 patients developed pan reactivity 1 patient died 5/6 required transfusion Hannon et al, 2014 Transfusion 54(2S):162A

21 POSSIBLE SOLUTIONS? 1. Dithiothreitol (DTT) treatment of red cells 2. Trypsin enzyme treatment of red cells 3. Cord cell panels 4. Neutralization of anti-cd38 5. Anti-CD38 antibody 6. Phenotyping/Genotyping

22 SOLUTION#1 - TREAT RED CELLS WITH DTT Denatures reagent red cell surface CD38 by disrupting disulfide bonds prevents dara binding However DTT also denatures other rbc antigens (e.g. Kell antigens) Anti-K is commonly encountered, significant antibody Need to provide K antigen negative units (unless patient known Kell positive)

23 Slide credit: Dr. Richard Kaufmann

24 BEST STUDY DTT VALIDATION Goal - determine if DTT method could be used to allow alloantibody identification in presence of dara Shipped two coded samples: DARA and DARA+AB Sent to 25 academic or reference labs along with instructions on how to perform DTT testing AB Screen Repeat Screen with DTT-RBC Identify AB with DTT- RBC Panel Chapuy et al, 2016 Transfusion doi:

25 BEST STUDY DTT VALIDATION RESULTS 25/25 found dara interference when testing with untreated rbc s Reactions strongest in solid phase 24/25 DTT treated screening cells eliminated positive reactions with Sample 1 (one site did not test due to negative screen with PEG on repeat testing) 25/25 had positive screens with Sample 2 using DTT-rbc. 100% of labs correctly identified antibody in Sample 2 with DTT-rbc with gel or tube Chapuy et al, 2016 Transfusion doi:

26 Chapuy et al, 2016 Transfusion doi:

27 DTT TREATMENT Fairly easy to use, widely available Inexpensive Must give K negative units Fails to detect antibodies to other DTT sensitive antigens: KEL, DO, IN, JMH, KN, LW Does not prevent future alloimmunization May not be practical for use in smaller blood banks

28 SOLUTION #2 - TRYPSIN ENZYME TREATMENT OF RBC Also cleaves CD38 from reagent red cells Inexpensive and fairly easily to use Can detect antibodies to Kell antigens However, less commonly used than DTT in blood banks Also cleaves antigens in other blood groups and will not detect antibodies to these antigens

29 SOLUTION #3 - CORD CELL ANTIBODY SCREEN Cord red cells lack or have very low CD38 Inexpensive, fairly easy to use, no treatment needed Limited commercially availability Potential to antigen type cord cells and use as panel for antibody detection. Could be stored frozen and thawed if needed. Fails to detect antibodies to other antigens not present/weakly expressed on cord red cells (Lewis, Rg, Ch, Sda, Yta, Xga)

30 SOLUTION #4 - NEUTRALIZATION WITH SOLUBLE CD38 Reagent red cells DARA CD38 Easy to use, all antibodies detected Commercially available, would likely work with all anti-cd38 Difficult to standardize amount of CD38 needed Expensive, short shelf life, not validated yet

31 SOLUTION #5 ANTI-CD38 IDIOTYPE ANTI-CD38 IDIOTYPE Easy, would not miss any antibodies Not currently available commercially Would require a different antibody for each anti- CD38 produced and knowledge of patient s medication history DARA

32 SOLUTION #6 - PHENOTYPE PATIENT Provide phenotype compatible units for Rh, K, Fy, Jk, S, s Also prophylactically avoids exposure to these antigens Needs to be done prior to starting anti-cd38 Cannot be done if transfused with red cells in past 3 months genotype instead Clinically significant antibodies can be missed (rare) depending on extent of matching AHG crossmatches with antigen matched units will still be incompatible

33 SOLUTION #6 - GENOTYPE PATIENT DNA based testing, TAT approx days Testing only needs to be done once Can be done after dara therapy started or if recently transfused Systems: Rh, Kell, Kidd, Duffy, MNS, Diego, Dombrock, Colton, Cartwright, Lutheran Expensive In rare cases, genotype does not accurate predict phenotype

34 ROLE OF PHENO/GENOTYPING IN TRANSFUSION MANAGEMENT Use of antigen matched units initially described for patients with warm autoantibodies Red cells provided based on extended pheno/genotype Provides blood safe for transfusion without adsorption techniques Also used for frequently transfused patients (SCD, Thal) Shirey et al Transfusion 2002: 42(11):1435

35 DOES DARA INTERFERE WITH OTHER LAB TESTS? Dara is an IgG kappa antibody Can be detected on SPE and IFE used to monitor M-protein levels May affect accurate assessment of response or disease progression in patients with IgG kappa myeloma protein McCudden et al, 2016 Clin Chem Lab Med: 54(6) 1095

36 ADDRESSING DARA INTERFERENCE Essential to include notification of transfusion service if patient receiving dara Interference identified early Complex investigations avoided Prevents delays in providing products

37 AABB RECOMMENDATIONS 1. BEFORE patient starts Dara: Baseline type and screen Phenotype/genotype 2. AFTER a patient starts Dara: ABO/RhD typing performed normally Antibody screen/identification completed with DTT-treated cells Provide K-negative units because DTT treatment destroys Kell antigen AABB Association Bulletin #16-02, January 2016

38 AABB RECOMMENDATIONS 3. For known alloantibodies, phenotypically/genotypically matched red cells may be provided. AHG crossmatches with these units will still be incompatible. 4. For emergency transfusion, uncrossmatched ABO, RhD compatible red cells can be given as per usual local practices. 5. Patients with negative antibody screen with DTT-treated panels, an electronic or IS crossmatch with ABO, RhD compatible, K-matched red cells can be performed. AABB Association Bulletin #16-02, January 2016

39 HOW IS UAH MANAGING DARA PATIENTS? CBS Lab notifying UAH blood bank of patients being started on daratumumab. Information added to UAH Blood Bank data system available in Edmonton Zone Phenotyping/genotyping performed Techs identify patients on DARA prior to starting extensive antibody investigation Patients provided with phenotypically matched red cells to reduce chances of reaction with underlying alloantibody and to help prevent future alloimmunization

40 OTHER POTENTIAL SITUATIONS Dara patient arrives at UAH without pheno/genotyping data available Unable to perform phenotyping due to recent red cell transfusion Patient requiring red cell transfusion urgently Possible solutions: Uncrossmatched ABO/Rh compatible rbc s still available in emergencies DTT available at CBS for further investigation

41 FUTURE CHALLENGES WITH mab S Monoclonal antibodies are novel class of therapeutics used for their specific targeting Many target antigens also expressed on red cells Potential for similar interferences with testing Trend to patients being treated earlier in disease, maybe discharged to management in community settings where testing capabilities may be limited.

42 QUICK RECAP Dara is a new treatment option in multiple myeloma Will likely continue to pose challenges in blood bank testing Dara interferes with pretransfusion blood bank testing mab s are novel treatment options in oncology Key element to managing this issue is identifying dara patients early and avoiding complex workups

43 Thank you