Those were the days…

Report
A candid retrospective of the
monoclonal revolution
Have the dreams come through ?
George Janossy
University College London
Nancy, 24th October 2008
A candid retrospective of the
monoclonal revolution
Have the dreams come through
?
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.
Dream No.1
Flow cytometry can be used for clinical
leukaemia diagnosis on the
‘immunophenotyping’ platform
Melvyn Greaves,
Geoff Brown and
George Janossy
anti-ALL (24 absorptions!)
fetal development:
BM is a lymphoid organ
TdT+,
cALL
(CD10)+
lymphoid
blast crisis
of Ph’+
chronic
myeloid
leukaemia
(Lancet ii: 1058,
1976)
The first clinical use of sorting in Europe
Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern
Cytomics Cytometry Part A 58A: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.
The extraordinary hypothesis by M.F.Greaves
leading to the immuno-phenotyping
 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 antiprecursor (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 immunohistochemistry (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 latetwentieth-century medicine. Cambridge: MIT Press; 2003.
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.
The very first immuno-histological preparation ever:
using monoclonal anti-human antibodies Oct.1978
NA1/34 CD1
monoclonal
antibody by
chicken
anti-human
Class II
McMichael &
Milstein
thymocyte – T lymphocyte maturation
Subcapsular
cortex
cortical
thymus
medullary
thymus
Intraepithelial lymphocytes
IgA
IgA
IgA
Selby et al.
Gut 22:169,1981,
CEI 44: 453. 1981
In the gut CD45+ intraepithelial lymphocytes (CD8+; not shown)
stained in combination with anti-IgA (lumen and plasma cells)
Janossy, G. et al. Nature 288: 81.1980
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
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?
b
a
BAL
PPD
24.2%
BAL
PPD
2.31%
IFN-g
2.48%
TNF-a
c
BAL
No Ag
d
0.78%
Blood
PPD
1.13%
IFN-g
35.1%
0.09%
0.02%
IFN-g
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
Dream No.3 – well, this is an ugly one
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

Panleucogating as the preferred method in resource-poor
countries Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-
Janossy G, Jani I, Gohde W. Affordable CD4(+) Tcell counts on 'single-platform' flow cytometers I. Primary CD4 gating. Br J
Haematol 2000; 111(4): 1198-208
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
 Monoclonal CD4 antibodies can be
How to cope
with THAT one?
Without going
bankrupt?
These samples
are from HIV+
patients waiting
for or on antiretroviral
therapy
See special
supplement
Clinical
Cytometry 74B;
2008.
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)
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
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.)

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).
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 immunotherapy

-, +/-, +, ++, +++ or negative, duller, dull, very weak,weak,a bit, medium, strong,
stronger, bright, brighter, mindboggling
Dream No. 5
 Monoclonal antibodies, humanized,
chimeric, complement-fixing or toxinlabelled, 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)

Integrated collaboration
Requires strong
leadership and a
long time-scale
(10-15 yrs)
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 cytometers as field instruments could
replace ELISA and RIA tests and are
also be capable of doing cellular
immunological tests such as CD4+ Tcell enumeration and Plasmodium
falciparum detection in whole blood.
Luminex-100; FACSArray with 96-well
microplate reader
Fig.2. The various applications of small, portable battery operated
clinical flow cytometers that operate on red diode laser include
haematological white blood cell counts and absolute CD4 counts [93], 5plex multiparameter analysis of immunoassays [this review] and the
analysis of parasitaemia during Plasmodium falciparum infection [94].
These instruments may establish a new field of flow cytometry that can
reach a wider range of laboratories than the current models.
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.
The first study
to show the
importance of
detecting
minimal
residual
leukaemia
Dream No.7
Salamanca
A.Orfao
Karolinska
A.PorwitMacDonald
St.Jude,
Memphis
D.Campana &
E. CoustanSmith
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
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.
MRD “Light” vs Standard MRD by Flow
Cytometry and PCR
Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102
Prognostic significance of CD19+ CD10+
and/or CD34+ at Day 19
Prognostic factor
P value*
CD19+ CD10+ and/or CD34+ (Present vs absent)
0.003
Age (<1 vs 1-9 vs >10 yrs)
0.906
WBC(<50 x 109/L vs ≥ 50 x 109/L)
0.095
DNA Index (≥1.16 vs others)
0.260
BCR-ABL
0.004
TEL-AML1
0.944
MLL-AF4
0.343
NCI Risk (Standard vs High)
0.342
*Univariate analysis; by multiple regression analysis MRD was the only significant
independent prognostic factor (P = 0.025)
Recife Pilot Study #1
Traditional
risk features
Day 19
CD19+ CD10+ and/or
CD34+
Risk
Group
Good
<0.01%
Low
Good
>0.01%
Standard
Poor
Any
High
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)
Results presented at SIOP, 2008
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
Brilliance, dedication and art
 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 hardearned 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
Grumbling about

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 FACSArray 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

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

Special issue of resource-restricted flow cytometr in Clinical Cytometry
Supplement 74B; 2008
Two models:
Central lab with
QA
Spreading its
influence to
daughter
laboratories
?
Simple
method in
the
‘bushes’
Can be complicated
and relatively
expensive,
Less expensive
Non-starter, only
good for wasting
the cash
Price tag $50-80/test
No FDA, only QA
Millions of $-s
FDA regulations may
‘secure’ complicated
protocols
Easy to train
Difficult to train the
beginners
Results can be
suboptimal, even in
the West
Simpler
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
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)
The best news for a while (EWGCCA)

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