Dual antibody immunohistochemistry: an efficient and sensitive tool for the detection of residual disease in chronic lymphocytic leukemia

  • Laura M WakeEmail author
  • Inhye E Ahn
  • Mohammed Z. Farooqui
  • Xin Tian
  • Maryalice Stetler-Stevenson
  • Gerald E. Marti
  • Adrian Wiestner
  • Irina Maric
Original Article


Highly effective treatments for chronic lymphocytic leukemia (CLL) have the potential to reduce significant tumor burden to single cells and therefore require sensitive tools to assess for minimal residual disease (MRD) in bone marrow (BM) biopsies. Flow cytometry (FC) is the current gold standard for detection of MRD, but requires a specialized facility, specific antibody panels, collecting hundreds of thousands-to-millions of cells, and personnel with expertise in the analysis and interpretation of FC MRD data, which may not be feasible in many small laboratories. Dual-antibody immunohistochemistry (DA-IHC) can identify abnormal populations better than morphology alone, but its correlation with FC assessment is not known. Our aims are to characterize the efficacy of DA-IHC in assessing BM samples post-treatment and to compare results with FC to evaluate for residual disease in CLL. We collected 2-year post-therapy data from 33 CLL patients on two treatment protocols, chemoimmunotherapy (CIT) and single agent ibrutinib (IB), as well as BMs from 10 healthy volunteers as morphologic controls. BM biopsy specimens were examined for the presence or absence of CLL based on morphologic evidence of lymphoid infiltration and aberrant co-expression of CD5 and PAX5 DA-IHC. FC using a standard CLL antibody panel was performed in parallel. All IB patients had residual disease detected by DA-IHC and FC, although five patients (22%) were morphologically negative by routine hematoxylin and eosin (H&E) stain. Those without overt morphologic evidence of disease showed DA-IHC-positive interstitial single cells and were positive for residual disease by FC. The assessments by DA-IHC and FC were significantly correlated (p = 0.004). Four patients (40%) treated with CIT were morphologically negative by H&E, and two of these had no detectable CLL by either DA-IHC or FC. The other two patients had low-level disease detected by both DA-IHC and FC. The MRD levels identified by DA-IHC were correlated with those by FC (p < 0.0001). We characterized the efficacy of DA-IHC in assessing CLL post-treatment and found assessments by DA-IHC and FC were statistically significant in both the IB- and CIT-treated patients. These findings suggest DA-IHC could accurately detect residual disease in cases lacking morphologic evidence by H&E alone. DA-IHC may be a useful tool in current practice as another sensitive and efficient method to assess for MRD in CLL.


Dual-antibody immunohistochemistry Chronic lymphocytic leukemia Minimal residual disease 


Compliance with ethical standards

Conflict of interest

Pharmacyclics and Novartis provided study drug and research support for the study. M.F. is employed at Merck & Co., Inc., Kenilworth, NJ, USA; owns stocks; and has received travel support from Merck. M.F. was an employee of the National Institutes of Health, Bethesda, MD, at the time of this study. A.W. received research support from Pharmacyclics, LLC, an AbbVie Company. Other authors declare that they have no conflict of interest.

Supplementary material

12308_2019_372_MOESM1_ESM.doc (69 kb)
ESM 1 (DOC 69 kb)
12308_2019_372_MOESM2_ESM.docx (124 kb)
ESM 2 (DOCX 124 kb)


  1. 1.
    Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H et al (2018) iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood. 131(25):2745–2760CrossRefGoogle Scholar
  2. 2.
    Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H et al (2008) Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 111(12):5446–5456CrossRefGoogle Scholar
  3. 3.
    Rawstron AC, Kennedy B, Evans PA, Davies FE, Richards SJ, Haynes AP et al (2001) Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood. 98(1):29–35CrossRefGoogle Scholar
  4. 4.
    Rawstron AC, Villamor N, Ritgen M, Bottcher S, Ghia P, Zehnder JL et al (2007) International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia. 21(5):956–964CrossRefGoogle Scholar
  5. 5.
    Hudnall SD, Ge Y, Wei L, Yang N-P, Wang H-Q, Chen T (2005) Distribution and phenotype of Epstein-Barr virus-infected cells in human pharyngeal tonsils. Mod Pathol 18(4):519–527CrossRefGoogle Scholar
  6. 6.
    Khokhar FA, Payne WD, Talwalkar SS, Jorgensen JL, Bueso-Ramos CE, Medeiros LJ, Vega F (2010) Angioimmunoblastic T-cell lymphoma in bone marrow: a morphologic and immunophenotypic study. Hum Pathol 41(1):79–87CrossRefGoogle Scholar
  7. 7.
    Masir N, Jones M, Lee AM, Goff LK, Clear AJ, Lister A, Marafioti T, Mason DY (2010) The expression of Bcl-2 by proliferating cells varies in different categories of B-cell lymphoma. Histopathology. 56(5):617–626CrossRefGoogle Scholar
  8. 8.
    Rimsza LM, Day WA, McGinn S, Pedata A, Natkunam Y, Warnke R, Cook JR, Marafioti T, Grogan TM (2014) Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas. Diagn Pathol 9:144CrossRefGoogle Scholar
  9. 9.
    Bottcher S, Ritgen M, Fischer K, Stilgenbauer S, Busch RM, Fingerle-Rowson G et al (2012) Minimal residual disease quantification is an independent predictor of progression-free and overall survival in chronic lymphocytic leukemia: a multivariate analysis from the randomized GCLLSG CLL8 trial. J Clin Oncol : Off J Am Soc Clin Oncol 30(9):980–988CrossRefGoogle Scholar
  10. 10.
    Santacruz R, Villamor N, Aymerich M, Martinez-Trillos A, Lopez C, Navarro A, Rozman M, Bea S, Royo C, Cazorla M, Colomer D, Gine E, Pinyol M, Puente XS, Lopez-Otin C, Campo E, Lopez-Guillermo A, Delgado J (2014) The prognostic impact of minimal residual disease in patients with chronic lymphocytic leukemia requiring first-line therapy. Haematologica. 99(5):873–880CrossRefGoogle Scholar
  11. 11.
    Kwok M, Rawstron AC, Varghese A, Evans PA, O’Connor SJ, Doughty C et al (2016) Minimal residual disease is an independent predictor for 10-year progression-free and overall survival in CLL. Blood. 128:2770–2773CrossRefGoogle Scholar
  12. 12.
    Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J, Hensel M, Hopfinger G, Hess G, von Grünhagen U, Bergmann M, Catalano J, Zinzani PL, Caligaris-Cappio F, Seymour JF, Berrebi A, Jäger U, Cazin B, Trneny M, Westermann A, Wendtner CM, Eichhorst BF, Staib P, Bühler A, Winkler D, Zenz T, Böttcher S, Ritgen M, Mendila M, Kneba M, Döhner H, Stilgenbauer S (2010) Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 376(9747):1164–1174CrossRefGoogle Scholar
  13. 13.
    Eichhorst B, Fink AM, Bahlo J, Busch R, Kovacs G, Maurer C, Lange E, Köppler H, Kiehl M, Sökler M, Schlag R, Vehling-Kaiser U, Köchling G, Plöger C, Gregor M, Plesner T, Trneny M, Fischer K, Döhner H, Kneba M, Wendtner CM, Klapper W, Kreuzer KA, Stilgenbauer S, Böttcher S, Hallek M, international group of investigators, German CLL Study Group (GCLLSG) (2016) First-line chemoimmunotherapy with bendamustine and rituximab versus fludarabine, cyclophosphamide, and rituximab in patients with advanced chronic lymphocytic leukaemia (CLL10): an international, open-label, randomised, phase 3, non-inferiority trial. Lancet Oncol 17(7):928–942CrossRefGoogle Scholar
  14. 14.
    Böttcher S (2014) Paving the road to MRD-guided treatment in CLL. Blood. 123(24):3683–3684CrossRefGoogle Scholar
  15. 15.
    Goede V, Fischer K, Busch R, Engelke A, Eichhorst B, Wendtner CM, Chagorova T, de la Serna J, Dilhuydy MS, Illmer T, Opat S, Owen CJ, Samoylova O, Kreuzer KA, Stilgenbauer S, Döhner H, Langerak AW, Ritgen M, Kneba M, Asikanius E, Humphrey K, Wenger M, Hallek M (2014) Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med 370(12):1101–1110CrossRefGoogle Scholar
  16. 16.
    Strati P, Keating MJ, O’Brien SM, Burger J, Ferrajoli A, Jain N, Tambaro FP, Estrov Z, Jorgensen J, Challagundla P, Faderl SH, Wierda WG (2014) Eradication of bone marrow minimal residual disease may prompt early treatment discontinuation in CLL. Blood. 123(24):3727–3732CrossRefGoogle Scholar
  17. 17.
    Molica S, Giannarelli D, Montserrat E (2019) Minimal residual disease and survival outcomes in patients with chronic lymphocytic leukemia: a systematic review and meta-analysis. Clin Lymphoma, Myeloma Leuk 19(7):423–430CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Pathology DepartmentJohns Hopkins MedicineBaltimoreUSA
  2. 2.Hematology Branch, National Heart, Lung and Blood InstituteNational Institutes of HealthBethesdaUSA
  3. 3.Office of Biostatistics Research, National Heart, Lung, and Blood InstituteNational Institutes of HealthBethesdaUSA
  4. 4.Laboratory of Pathology, National Cancer InstituteNational Institutes of HealthBethesdaUSA
  5. 5.Department of Laboratory MedicineNational Institutes of HealthBethesdaUSA

Personalised recommendations