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A candid retrospective of the monoclonal revolution Have the dreams come through ? George Janossy

A candid retrospective of the monoclonal revolution Have the dreams come through ? George Janossy University College London. Nancy, 24 th October 2008. A candid retrospective of the monoclonal revolution Have the dreams come through ?.

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A candid retrospective of the monoclonal revolution Have the dreams come through ? George Janossy

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  1. A candid retrospective of the monoclonal revolution Have the dreams come through ? George Janossy University College London Nancy, 24th October 2008

  2. A candid retrospective of the monoclonal revolution Have the dreams come through ?

  3. The shepperd falls asleep at the camp-fire -- with fabolous consequences. He has the dream of the dreams: dozens of diamonds are sparkling up in the flames. When he wakes up many of the these dream-diamonds vanish -- but …. But miracolously, seven of these gems still remain ! This talk is about these seven wonderous dream-diamonds.

  4. Flow cytometry can be used for clinical leukaemia diagnosis on the ‘immunophenotyping’ platform anti-ALL (24 absorptions!) fetal development: BM is a lymphoid organ Dream No.1 Melvyn Greaves, Geoff Brown and George Janossy

  5. TdT+, cALL (CD10)+ lymphoid blast crisis of Ph’+ chronic myeloid leukaemia (Lancet ii: 1058, 1976)

  6. The first clinical use of sorting in Europe Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Cytomics Cytometry Part A58A:87–97 (2004). Janossy G, Francis GE, Capellaro D, Goldstone AH, Greaves MF. Cell sorter analysis of leukaemia-associated antigens on human myeloid precursors. Nature 1978; 276(5684): 176-8.

  7. Acute lymphoid leukaemia (ALL) must come from an early stem cell – which will have surface markers these will not be leukaemia specific: it will be the first anti- precursor (stem) cell antiserum balanced experimental design T heterologous antiserum (absorbed, specific) B surface Ig and Class-II (absorbed, specific) myeloid markers all this on leukaemias and normal cells Terminal transferase (Hoffbrand, Bollum) and immuno-histochemistry (Catovsky, D.Y.Mason) Cell lines (Minowada) Fluorescence-activated cell analysis and sorting (FACS) THE PLATFORM WAS BORNE; see GEIL, EuroFlow, etc. reviewed by Keating P Cambrosio A. Biomedical platforms—realigning the normal and the pathological in late-twentieth-century medicine. Cambridge: MIT Press; 2003. The extraordinary hypothesis by M.F.Greavesleading to the immuno-phenotyping

  8. Dream No.2 Monoclonal antibodies can be used for visualizing cells in tissues, normal and pathological (‘monoclonal immunohistology’ platform) CD1 (NA1/34) on human thymus: The first ever tissue section stained by the first anti-human monoclonal antibody CD1 (NA1-34) and CD45 (2D1) for T-ALL diagnosis and thymic origin Ken Bradstock, Gianni Pizzolo Bradstock KF, Janossy G, Bollum FJ, Milstein C. Anomalous phenotype in thymic acute lymphoblastic leukaemia. Nature 1980; 284(5755): 455-7.

  9. thymocyte – T lymphocyte maturation Subcapsular cortical medullary cortex thymus thymus The very first immuno-histological preparation ever: using monoclonal anti-human antibodies Oct.1978 NA1/34 CD1 monoclonal antibody by McMichael & Milstein chicken anti-human Class II

  10. Intraepithelial lymphocytes IgA IgA IgA Selby et al. Gut 22:169,1981, CEI 44: 453. 1981 In the gutCD45+ intraepithelial lymphocytes(CD8+; not shown) stained in combination with anti-IgA(lumen and plasma cells)

  11. Janossy, G. et al. Nature 288: 81.1980

  12. This is, however, old-fashioned amateur work when compared to the informative power and clarity of confocal microscopy and to the elegance of dynamic intravital imaging ( Jackson Eger; this meeting) This latter work also brings up considerations about lymphocytes migrating to sites, and slowing down there, where the antigens are deposited: for diagnosing active tuberculosis by flow cytometry using sputum-borne lymphocytes

  13. b a BAL BAL 35.1% PPD PPD 24.2% 2.31% 2.48% g a IFN - TNF - c d Blood BAL PPD 0.09% No Ag 0.78% 1.13% 0.02% CD 4 g g IFN - IFN - Flow cytometry of active tuberculosis Broncho-alveolar lavage (blood versus lung) -- paper by S.Barry et al. JCI 2004) TB is a lung-disease Analysis of IFN-g synthetic capacity of lung-derived lymphocytes: What do you think to be the more informative technology: Flow-cytometry or ELISpot? Janossy, G. et al.: The role of flow cytometry in the IFN-g based diagnosis of active tuberculosis and its coinfection with HIV-1 – a technically oriented review. Cytometry Part B 74B (suppl.1) S141-151

  14. Dream No.3 – well, this is an ugly one • Monoclonal CD4 antibodies can be used to monitor patients: ‘slope’ of progression to AIDS and CD4 regeneration on therapy (‘primary CD4 gating’) • Documenting the natural history of HIV disease in the Royal Free Haemophilia cohort (from 1982 CD4 counting onwards) Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, Kernoff PB. Serial CD4 lymphocyte counts and development of AIDS. Lancet 1991; 337(8738): 389-92. • Primary CD4 gating Janossy G, Jani I, Gohde W. Affordable CD4(+) T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating. Br J Haematol 2000; 111(4): 1198-208 • Panleucogating as the preferred method in resource-poor countries Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. Cytometry 2002; 50(2): 69-77. Also: Glencross et al. Cytometry Part B &4B S40-S51 CD4 – CD8

  15. How to cope with THAT one? Without going bankrupt? These samples are from HIV+ patients waiting for or on anti-retroviral therapy See special supplement Clinical Cytometry 74B; 2008.

  16. Glencross D, Aggett H, Stevens WS and Mandy F.: African Regional external Quality Assessment for CD4 evaluation, and performance of laboratories. Clinical Cytometry 74B: S69-79. (2008)

  17. Flow cytometry is quantitative quantitative: the optimal method to count, precisely and accurately, large numbers of cells in large numbers of samples (absolute numbers and percentages of cells) the next topic is to show that: quantitative: the optimal method to count, precisely and accurately, the numbers of molecules on the cell surfaces – in correlation with the mean fluorescence intensity (MFI) this works best with FITC and PE – and not so much with the tandem flourochrome conjugated markers flow cytometry can be contained to remain simple and it beats other methods, such as ELISpot, hands down in terms of quantitative potentials

  18. Dream No. 4 It is so easy to transform the values of mean fluorescence intensity (MFI) into values of expressed molecules per cells because reliable method such as the QIFI (Quantitative Indirect Immuno-Fluorescence) exists and the biological controls are so stable CD4 on T cells, CD45 on lymphocytes show virtually no variations; experienced flow cytometrists are constantly using these normal controls and many others to standard runs on their cytometers (Carleton Steward et al.)

  19. A range of standard values (QIFI) Bikoue A, George F, Poncelet P, Mutin M, Janossy G, Sampol J. Quantitative analysis of leukocyte membrane antigen expression: normal adult values. Cytometry 1996;26:137–147.Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Cytomics Cytometry Part A 58A:87–97 (2004).

  20. The clinical use of quantitative values for MFI unusual populations clear subsets definition of cut-off points to define subsets aberrant combinations of markers leukaemias, lymphomas alterations due to immuno- therapy -, +/-, +, ++, +++ or negative, duller, dull, very weak,weak,a bit, medium, strong, stronger, bright, brighter, mindboggling

  21. Dream No. 5 Monoclonal antibodies, humanized, chimeric, complement-fixing or toxin-labelled, are going to be therapeutically useful in collaboration with Sandoz/Novartis; the third successful therapeutic agent were made (Janossy, Amlot: basilimaximab – Simulect: anti-CD25 to ameliorate organ rejection given together with Cyclosporin) Ricin-A chain conjugated CD22 (in collaboration with E. Vitetta and P. Amlot)

  22. Integrated collaboration Requires strong leadership and a long time-scale (10-15 yrs)

  23. Dream No. 6: “one-stop shop” An emerging technology is the multiplexed immunoassays read by flow cytometry, for the diagnosis of infectious diseases. In these assays, multiple fluorescent microspheres, conjugated to different antigens or antibodies, constitute the solid phase for detecting antibodies or antigens in biological samples. These flow cyto-meters as field instruments could replace ELISA and RIA tests and are also be capable of doing cellular immunological tests such as CD4+ T-cell enumeration and Plasmodiumfalciparum detection in whole blood. Jani IV, Janossy G, Brown DW, Mandy F. Multiplexed immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor settings. Lancet Infect Dis 2002; 2(4): 243-50. Luminex-100; FACSArray with 96-well microplate reader

  24. The first study to show the importance of detecting minimal residual leukaemia Salamanca A.Orfao Karolinska A.Porwit-MacDonald Dream No.7 St.Jude, Memphis D.Campana & E. Coustan-Smith

  25. On therapy, normal B-cell precursors are temporarily absent Aberrant combinations of markers can identify malignant cells, even if present in very low proportions in the sample. The induction-therapy produced effects on the normal populations provides chances for simplifying the assays This arrangement is taking the the parameter of ‘blast decrease rate’ directly relevant to patient management bone marrow samples 18-22 days post-induction therapy

  26. Minimal Residual Leukaemic Disease (MRD) ‘lite’ version This was developed by Campana and Coustan-Smith at St.Jude, Memphis, and then formed the diagnostic basis for ‘out-sourcing’ from St.Jude to Brasil, the poor area of the country such as Recife. T.Eden and others called attention that many ALL-sufferer children were lost to therapy by toxicity, abandonment and secondary infections. So, good responders get less drugs. Sandlund et al. Blood 2002: 100: 43-47. Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102. Howard S C, Pedrosa M et al. JAMA 2004; 291: 2451-75. Howard SC, Campana D et al. Leukemia 2005; 19: 323-5. Reviewed by Janossy G. The changing pattern of smart flow cytometry Biotechnology J. 3: 32-42. 2008.

  27. MRD “Light” vs Standard MRD by Flow Cytometry and PCR Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102

  28. Prognostic significance of CD19+ CD10+ and/or CD34+ at Day 19 *Univariate analysis; by multiple regression analysis MRD was the only significant independent prognostic factor (P = 0.025)

  29. Recife Pilot Study #1 Poor = T-lineage ALL, or B-lineage ALL with WBC ≥ 50K, age 10-15 yrs, testicular/CNS leukemia, and/or adverse genotypes (BCR-ABL, MLL rearrangements, hypodiploidy <45 chromosomes)

  30. REFINING AND REDUCING INTENSITY OF THERAPY FOR PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) IN DEVELOPING COUNTRIES: THE RECIFE-ALL-2005 STUDY Pedrosa F 1, Rivera GK2, Campana D2, Coustan-Smith E2 , Pedrosa M1, Lins M1, and Ribeiro RC2 1Instituto Materno Infantil de Pernambuco, Recife, Brazil and 2 St. Jude Children’s Research Hospital, Memphis, TN, USA Results presented at SIOP, 2008

  31. That is flow cytometry – a collaborative, constantly developing practical science with academic involvement (could be stronger) and loyal and efficient industry support (who are not charitable institutions and are entitled to their hard-earned profits) It is a quantitative method to count cells and quantitative to count molecules this lecturer is a happy person (xtatic) is then there anything to grumble about ? well, if you insist, there are two problems Brilliance, dedication and art

  32. Flow cytometry could be far more powerful; it could reach areas other techniques can not (Heineken beer effect) Methods could be introduced faster How long are we going to wait to introduce CD45 for quality controlling haematological differential counts? What is the case with paediatric infectious disease diagnosis ? Why “dream 6 (one-stop shop)” is not coming along when the best instruments such as the Luminex-100 and the FACS-Array are all there ready to be used ? How is it possible the ELISpot for tuberculosis diagnosis is spreading (non-quantitative and inflexible method) and flow cytometry fails to make a proper impact ? Grumbling about

  33. The flow cytometry with its enthusiastic supporting science provides the impression to the ‘uninitiated’ (who are…) that it is over-complicated and frequently simply incomprehensible please, once a method is introduced and works, commit yourself to a final stage of simplification and explain this to the outside crowd please, trust that you technology is simply unbeatable even if it is explained well please, do not over-spend – hard times are here and are still coming Grumbling about Special issue of resource-restricted flow cytometr in Clinical Cytometry Supplement 74B; 2008

  34. Two models: ? Central lab with QA Simple method in the ‘bushes’ Spreading its influence to daughter laboratories Can be complicated and relatively expensive, Price tag $50-80/test FDA regulations may ‘secure’ complicated protocols Difficult to train the beginners Results can be suboptimal, even in the West Non-starter, only good for wasting the cash Millions of $-s Simpler Less expensive No FDA, only QA Easy to train Better results than the West high volume flow cytometry for huge numbers Larsen,C.H: The fragile environments of inexpensive CD4+ T cell counting in the least developed countries Clin.Cytometry, 73B: Jan. 2008 ‘smart’ flow cytometry (Janossy, G. Biotechn. J. 2008; 3: 32-42

  35. Flow Cytometry for the Global Village: Organized quality assessment (QA) schemes are essential for training and education (QASI, NEQAS and External QA in the Netherlands)

  36. The best news for a while (EWGCCA)

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