Leukemia(1995) 10, 1648-1652
`e 1996 Stockton Press Allrights reserved 0887-6924/96 $12.00
`
`OPEN FORUM
`
`syndromes
`in myelodysplastic
`A paradigm shift
`A Raze". S Mundie". V Shettv', S Alvi", H Chopra", L Span', A Parcharldou",
`S Dar". P Venugopal'.
`J Showel",
`J Lcew", E Robin", S Rifkin", D Alston", B Hernandez!'>, R Shah", H Kaizer". S Gregory'
`
`R Borck", S Gezer',
`and H Preisler'
`
`1 Rush Cancer Institule, Rush-Presbyterian-St Luke's Medical Center, Chicago, IL, USA; 22nd Department of Internal Medicine, University of
`Athens, Hospital Evangelismos, Athens, Greece; 3Department of Pathology, Rush-Presbyterian-St Luke's Medical Center, Chicago, IL; 4/ngalf's
`Memorial Hospital, Harvey, IL, "Nortnwest Community Hospital, Arlington Heights, fL, »terone Cancer Treatment Center, LaPorte, IN, and
`7Lake Forest Hospital, Deerfield, IL, USA
`
`treatment
`and rational
`objective
`with the hope of developing
`approaches
`assigned to reverse the lesion(s). Our own
`studies
`the last 10 years
`combined
`with
`those of others
`over
`have
`allowed
`us to develop
`a new model
`for
`the pathogenesis
`of
`MDS which we believe
`constitutes
`a paradigm shift
`in these
`disorders.
`The purpose
`of
`this article
`is to summarize
`these
`studies and present
`the new paradigm.
`
`MDS is a stem cell disease
`
`have
`studies
`and molecular
`biochemical
`sophisticated
`Highly
`the bone marrow in MDS pati-
`repeatedly
`demonstrated
`that
`ents is monoclonal.
`'8-2) This monoclonality
`should
`not
`be
`confused with the monoclonality
`of malignancy.
`For example,
`de novo standard
`risk primary
`acute myeloid
`leukemia
`(AMU
`disease, but here the clonalitv refers to only
`is a monoclonal
`the leukemic
`cells or blasts, while
`the residual
`hematopoietic
`not clonal." In MDS on the other hand,
`cells are usually
`8M consists
`of cells
`belonging
`to erythroid,
`myeloid
`the
`megakaryocytic
`lineages
`(as well
`as
`probably
`lymphocytes)
`at all stages of differentiation
`(albeit dysplastic)
`but all of which
`are the descendants
`of one single diseased
`parent stem cell.
`In other words,
`the transforming
`event
`in the
`stem cell
`conferred
`upon
`it a growth
`advantage
`so that
`the
`entire 'feeder'
`compartment
`of pluripotential
`cells was over-
`whelmed
`by the progeny
`of the transformed
`cell
`(see Figure
`1).
`In this sense, MDS is closer
`to the chronic
`phase of chronic
`myeloid
`leukemia
`(CML) where
`the (normally)
`differentiated
`
`the
`and
`B
`
`the pluripoten-
`event(s) affects
`defined transforming
`A poorly
`In myelodysplastic
`syndromes
`tial bone marrow (BM) stem cell
`(MOS),
`'conferring
`a growth
`advantage
`upon
`It Which leads
`eventually
`to monoclonal
`hematopoiesis.
`The progeny
`of
`this
`transformed
`ancestor
`undergo
`recognizable
`albeit dysplastic
`maturation. We propose that
`this picture is further
`complicated
`by a variety of cytokines,
`tumor necrosis
`factor alpha (TNF-a),
`1(3 (ILa
`transforming
`growth factor beta (TGF-13) and Interleukln
`1(3) which exert a dual effect on the diseased cells. The imma-
`ture C034+ cells are stimulated
`to proliferate, while their
`later
`differentiated
`daughters
`are induced
`to undergo
`apoptosis
`accounting
`the clinical
`syndrome
`of pancytopenia
`despite
`for
`hypercelluJar
`BMs. Studies 'directed
`at measuring
`the rates of
`proliferation
`and apoptosis
`as well as the levels of TNF-a, TGF-
`fJ and IL-1Jl confirm this
`hypothesis
`and are presented
`in
`greater detail. A novel approach towards MDS therapy emerges
`as a result of
`this paradigm shift based upon the premise
`that
`antl-cytoktne
`therapy would prevent excessive
`intramedullary
`apoptosis
`and result
`in improved
`cytopenlas
`as well as cause
`a slowing
`down of
`the diseased
`precursor
`cell proliferation
`resulting
`in resumption
`of polyclonal
`hematopoiesis.
`Because
`a number of cytokines
`function
`through
`common
`lipid second
`messengers,
`interruption
`of
`this pathway
`should
`theoretically
`of a cascade of cytokines.
`cause disruption
`In the signalling
`Keywords:
`rnyelodysplastic
`syndromes (MDS);
`tumor necrosis fac-
`tor alpha (TNF-a);
`transforming growth factor beta (TGF-f3);
`interleu-
`kin 1~ (IL-1m; apoptosis
`
`Introduction
`
`(MDS) are indolent
`dis-
`donal
`syndromes
`The myelodysplastic
`orders which
`predominate
`in the elderly
`and which
`clearly
`involve
`a diseased
`pluripotential
`hematopoietic
`stem cell
`since monoclonality
`of at
`least
`the non-lymphoid
`cells is the
`rule."? The clinical
`presentation
`poses an apparent
`paradox
`in
`that
`the majority
`of patients
`have variable
`cytopenias
`despite
`30% patients
`hypercellular
`marrows.
`Approximately
`evolve
`into acute leukemia which
`is commonly
`of
`the myeloid
`var-
`iety, although
`lymphoid
`transformation
`has also been clearly,
`rarely, demonstrated
`in these patients.s-t'' Abnormalities
`albeit
`by
`of chromosomes
`5 and 7 are rather
`common
`followed
`8, 20 and 17.11-14 The only
`those affecting
`chromosomes
`treatment
`option
`for
`the majority
`of MDS patients
`continues
`to be supportive
`care since various
`therapeutic
`approaches
`ranging from the use of vitamins
`and growth
`factors
`to inten-
`sive chemotherapy
`have failed to benefit a substantial
`number
`of patients.'>!" Given this depressing clinical
`scenario,
`efforts
`have been focused at understanding
`the biology
`of MDS better
`
`~-c~~_
`~;.(it~
`'-1 ...
`'~@
`
`Progenitor
`
`\
`
`5. ~20"
`-7
`
`Sleeper /
`
`Feeder
`
`)
`
`Commlttlrd lymphoid
`
`-s:
`
`• •I
`
`o 00o 0
`
`000
`
`•
`
`~ l.
`o
`Differentiated 0 000 00
`cells
`_.. 00
`0
`o
`
`Correspondence: A Raza, Leukemia Program, Rush-Presbvterian-St
`Luke's Medical Center, 2242 West Harrison Street, Suite 108,
`lllinols 60612, USA
`Chicago,
`Received 22 April 1996; accepted 17 June 1996
`
`Killman's model of normal hematopoiesis has been util-
`Figure 1
`ized to describe the paradigm shift
`in myelodysplastic
`syndromes.
`Note that the 'initial event(s)'
`in MDS affects a stem cell
`in the 'feeder'
`compartment while commonly encou-ntered cytogenetic abnormali-
`ties affecting chromosome 5, 7, 8 etc are later evolutionary
`events.
`
`DR. REDDY’S LABS., INC. EX. 1019 PAGE 1
`
`

`

`erythroid, myeloid and megakaryocytic cells are all marked
`by the pathognomonic Philadelphia (Ph') chromosome."
`In
`CML, the initial
`'event' appears to be the Ph' translocation
`since the BM resumes a polyclonal nature upon disappear-
`ance of this cytogenetic abnormality in response to interferon
`therapy."
`In MDS on the other hand, cytogenetic abnormali-
`ties such as those affecting chromosomes 5 or 7 appear to be
`epiphenomena or secondary events since they often involve
`a proportion of cells in a marrow which is otherwise mono-
`In MDS therefore,
`the first consequence of
`the
`clonal.11,26
`transforming event is monoclonality and cytogenetic abnor-
`malities represent disease evolution while in CML,
`the Ph'
`translocation may be the 'transforming event' since there is
`no evidence of monoclonal hematopoiesis upon eradication
`of the Ph' chromosome. The model of normal hematopoiesis
`proposed by Killman"
`is particularly relevant
`in this setting
`(Figure 1).
`
`Nature of the transforming event
`in MDS
`
`is obscure and ambiguous
`
`Since there are certain cytogenetic abnormalities which occur
`in MOS with a regular frequency,
`it
`is not unreasonable to
`look for genic abnormalities associated with the most com-
`monly involved areas of the affected chromosomes.
`In MOS,
`the interstitial deletion involving 5q31.1 with its associated
`loss of the IRF-l gene is one such abnormality." The 5q31.1
`deletion is present in 30% MDS patients and 50% AML pati-
`entswho developed leukemia secondary to a preceding mye-
`Jcdvsplasia."
`Located at
`this
`site is the transcription
`enhancer/tumor suppressor IRF-l gene" which can also be
`functionally
`inactive due to accelerated -exon skipping'
`despite the presence of 3. normal gene.'? It
`is also possible
`that exaggerated exon skipping in the allele containing the
`normal
`IRF-l superimposed upon the deleted IRF-l allele
`accounts for the evolution of MDS to AMl. Conversely, over-
`expression of
`IRF-2 can suppress the effects of IRF_l.31 The
`problem with implicating the IRF-l
`in the 'transforming event'
`isthat this deletion was detected in only 20-80% of the hema-
`topoietic cells in MDS patients correlating with the percentage
`of blasts'" rather than the 100% of the cells expected on the
`basisof the monoclonal nature of the disease. In other words,
`I a genetic abnormality which only affects a proportion of the
`MDS cells instead of all
`the monoclonal cells by definition
`cannot be a candidate for the transforming event.
`Inappropriate expression of another gene,
`the esotropic
`virus integration site 1 gene (Evi-l) associated with retroviralfy
`induced myeloid leukemias in mice32 has also been associ-
`ated with AML and MDS in humans." Aberrant expression of
`Evi~1 conferred a non-responsiveness in murine BM progeni-
`tor cells to the effects of erythropoietin, an observation parti-
`cularly relevant to MDS since the erythroid cells appear to be
`universally most diseased in these disorders. Because abnor-
`malitiesof Evi-l are not found in all MDS patients, once again
`it is difficult to consider this gene as being somehow involved
`in the initial
`'transforming event'. Other molecular
`lesions
`which have been described include mutations of ras.>' fms,35
`p5336 as well as deregulation of other as yet unidentified
`tumor suppressorgenes."
`In summary therefore, MDS are clearly stem cell disorders
`althoughthe nature of the initial event is unknown. Given that
`thesecomplex-syndromes are highly heterogeneous in their
`clinical presentations, it is likely that the initial event(s) will
`from one group of
`I parallel this heterogeneity-being different
`
`\
`
`1649
`
`Open forum
`
`1
`
`if not varying amongst
`individuals.
`MDS patients to another,
`Whatever the initial event
`is in MOS
`its consequences are
`very well appreciated. The affected cell develops a growth
`advantage over
`its neighbors eventually
`leading to mono-
`clonal hematopoiesis. Once the entire 8M is descending from
`a single parent cell,
`the likelihood of additional mutations
`accumulating in one of
`its daughters is very great. Further,
`the evolved and additionally mutated cell will have a greater
`chance of survival and future proliferation in a monoclonal
`setting as compared to a polydonal one because all the sur-
`rounding sister cells share in the same 'initial evenf and thus
`are not very different
`from it. The expansion (and perhaps
`further mutations) of one such clone is probably responsible
`for the eventual evolution to acute leukemia. The key conse-
`quence of the stem cell defect in MDS then is monoclonalitv
`which predisposes these patients to frank malignancy.
`In fact,
`the definition of pre-leukemia should perhaps be rendered
`more objective by restricting the term to individuals in whom
`monoclonality
`(in the absence of a substantial number of
`blasts) can be documented.
`
`What causes the clinical syndrome of MDS?
`
`The apparent paradox of variable cytopenias in the presence
`of generally cellular marrows in these patients could be
`accounted for by increased proliferation
`of hematopoietic
`cells being cancelled by an equally increased rate of intrame-
`dullary apoptosis. Thus, despite frantic activity in the marrow
`compartment,
`the peripheral blood would remain devoid of
`functional
`hematopoietic
`cells.
`In fact, our own studies
`directed at measuring the in vivo rates of proliferation and
`programmed cell death have demonstrated the above hypoth-
`esis to be quite true. By infusing MDS patients with thymidine
`analogues iodo- and/or bromodeoxyuridine (lUdR, BrdU) as
`previously descnbed.w-" we found that
`the total cell cycle
`time (Tc) in 120 MDS patients was a median of 35 h while
`the labeling index (L1)was 26.1%. Figure 2 shows a double-
`labeled slide in an MDS patient where large numbers of cells
`can be seen synthesizing DNA. Thus, MDS are clearly actively
`proliferative disorders with no dearth of S-phase cells in their
`bone marrows. Unfortunately,
`the rapid proliferation is coun-
`tered by rapid and excessive cell death.
`
`~."
`
`.....
`
`'0-:'
`
`-'
`
`...•...$
`
`Double labeling for the two thymidine analogues iodo-
`Figure 2
`deoxyuridine (lUdR) (brown) and bromodeoxyuridine
`(BrdU) (blue)
`depicting the cells synthesizing DNA in the bone marrow biopsy of
`an MDS patient. Original magnification x 400.
`
`DR. REDDY’S LABS., INC. EX. 1019 PAGE 2
`
`

`

`leukemia/1996J 10, 1648-1652
`© 1996 Stockton Press Allrights reserved 0887-6924/96 $12.00
`
`OPEN FORUM
`
`A paradigm shift
`
`in myelodysplastic
`
`syndromes
`
`A Raza'. 5 Mundle1
`S Dar". P Venugopal'.
`, V Shettv', 5 Alvi', H Chopra", L Span'. A Parchartdou-,
`R Borok ', 5 Gezer",
`J Showell,
`J Loew', E Robin", S Rifkin", D Alston", B Hernandez", R Shah", H Kaizer", 5 Gregory'
`and H Preisler1
`1 Rush Cancer tnsttune, Rush-Presbyterian-St Luke's Medical Center, Chicago, IL, USA; 22nd Department of Internal Medicin~, University of
`Athens; Hospital Evangelismos, Athens, Greece; "Depestmem of Pathology, Rush-Presbyterian-St Luke's Medical Center, Chicago, IL; 4/ngaJl's
`Meriwrial Hospital, Harvey, IL, "Northwest Community Hospital, Arlington Heights, IL, fiLaPorteCancer Treatment Center, LaPorte, IN, and
`7 Lake Forest Hospital, Deerfield, IL, USA
`
`treatment
`and rational
`objective
`with the hope of developing
`assigned to reverse the lesionrs). Our own studies
`approaches
`the last 10 years
`combined
`with
`those of others
`over
`have
`of
`allowed
`us to develop
`a new model
`for
`the pathogenesis
`MDS which we believe
`constitutes
`a paradigm shift
`in these
`disorders.
`The purpose
`of
`this article
`is to summarize
`these
`studies and present
`the new paradigm.
`
`MDS is a stern cell disease
`
`j
`have
`studies
`and molecular
`biochemical
`sophisticated
`Highly
`I
`the bone marrow in MDS pati-
`repeatedly
`demonstrated
`that
`ents is rnonoclonal.w-"
`not be }
`This monodonality
`should
`1
`confused with the monodonality
`of malignancy.
`For example,
`de novo standard
`(AML)
`risk primary
`acute myeloid
`leukemia
`disease, but here the clcnatftv refers to only
`is a monoclonal
`the leukemic
`cells or blasts, while
`the residual
`hematopoietic
`not clonal.> In MDS on the other
`cells are usually
`hand,
`BM consists
`of cells
`belonging
`to erythroid,
`myeloid
`the
`megakaryocytic
`lineages
`(as well
`as
`probably
`lymphocytes)
`at all stages of differentiation
`(albeit dysplastic)
`but all of which
`are the descendants
`of one single
`diseased
`parent stem 'cell.
`In other words,
`the transforming
`event
`in the
`stem cell
`conferred
`upon
`it a growth
`advantage
`so that
`the
`of pl uri potential
`cells was over-
`entire
`'feeder'
`compartment
`whelmed
`by the progeny of the transformed
`cell
`(see Figure 1).
`In-this sense, MDS is closer
`to the chronic
`phase of chronic
`myeloid
`leukemia
`(CML) where
`the (normally)
`differentiated
`
`r
`
`I.
`
`.\
`)
`
`the
`and
`B
`
`Feeder
`
`Sleeper
`
`the pluripoten-
`event{s) affects
`A poorly defined transforming
`in myelodysplastic
`syndromes
`tial bone marrow (BM) stem cell
`(MOS),
`'conferring
`a growth
`advantage
`upon
`it which
`leads
`eventually
`to monoclonal
`hematopoiesis.
`The progeny
`of
`this
`transformed
`ancestor
`undergo
`recognizable
`albeit dysplastic
`maturation. We propose that
`this picture is further
`complicated
`by a variety of cytokines,
`tumor necrosis
`factor alpha (TNF-O'),
`113 (IL-
`transforming
`growth factor beta (TGF-p) and interleukin
`1p) which exert a dual effect on the diseased
`cells. The imma-
`ture C034+ cells are -stlmulated
`to proliferate,
`while their
`later
`differentiated
`daughters
`are induced
`to undergo
`apoptosis
`accounting
`the clinical
`syndrome
`of pancytopenia.
`despite
`for
`hypercellillar
`BMs. Studies "directed at measuring
`the rates of
`proliferation
`and apoptosis
`as well as the levels of TNF-a:, TGF-
`f3 and,
`IL-1JJ confirm this
`hypothesis
`and are presented
`in
`greater detail. A novel approach towards MeS therapy emerges
`as a result of this paradigm shift based upon the premise that
`antl-cytcklne
`therapy, would
`prevent excessive
`intramedullary
`apoptosls
`and result
`in improved
`cytopentas
`as well as' cause
`a slowing' down
`of
`the diseased
`precursor
`cell proliferation
`resultlnq
`in resumption
`of polyclonal
`hematopoiesis.
`Because
`a number. of cytokines
`function
`through
`common
`lipid second
`messengers,
`Interruption
`of
`this pathway
`should
`theoretically
`cause disruption
`in the signalling
`of a cascade of cytokines.
`Keywords: myelodysplastic
`syndromes (MDS);
`tumor necrosis fac-
`tor alpha (TNF-a);
`transforming growth factor beta (TGF·j3);
`interieu-:
`kin 1~ (IL-l~),apoptosis
`.
`
`Introduction
`
`clonal dis-
`(MDS) are indolent
`The mvelodysplastic
`syndromes
`orders which
`predominate
`in the elderly
`and which
`clearly
`involve
`a diseased
`pi uri potential
`hematopoietic
`stem cell
`since monoclonality
`of at
`least
`the non-lymphoid
`cells is the
`rule.'-" The clinical
`presentation
`poses an apparent
`paradox
`in
`that
`the majority
`of patients
`have variable
`cytopenias
`despite
`hypercellular
`marrows.
`Approximately
`30% patients
`evolve
`the myeloid var-
`into acute leukemia which
`is commonly
`of
`iety, although
`lymphoid
`transformation
`has also been clearly,
`in these patlcrrrs.s-tv Abnormalities
`albeit
`rarely, demonstrated
`5 and 7 are rather
`by
`of chromosomes
`common
`followed
`8, 20 and 17.11-14 The only
`those
`affecting
`chromosomes
`of MDS patients
`treatment
`option
`the majority
`continues
`for
`to be supportive
`care since various
`therapeutic
`approaches
`ranging from the use of vitamins
`and growth
`factors to inten-
`sive chemotherapy
`have failed to benefit a substantial
`number
`of patfcnrs.v-t>
`Given this depressing
`clinical
`scenario,
`efforts
`have been focused
`at understanding
`the biology
`of MDS better
`
`r ~-c~.._
`#"*~\;-
`\" -i ".
`-1i.~'.
`• •j° 00o 0
`
`Progenitor
`
`Committed Lymphoid
`Progenitor
`
`00 °
`
`o
`. Differentiated 0. 000 00
`cells
`o
`$
`.00
`o
`
`.
`
`'j
`1
`J
`"
`
`I
`"
`,
`
`Correspondence: A Raza, Leukemla Program, Rush-Presbvterian-St
`luke's Medical Center, 2242 West Harrison Street, Suite 108,
`Chicago,
`Illinois 60612, USA
`Received 22 April 1996; accepted l Zfune 1996
`
`Killman's model of normal hematopoiesis has been util-
`Figure 1
`ized to describe the paradigm shift
`in myelodysplastic
`syndromes.
`Note that the 'initial eventts)'
`in MDS affects a stem cell
`in the 'feeder'
`compartment while commonly encounfered cytogenetic abnormal
`i-
`ties affecting chromosome 5, 7, 8 etc are late, evolutionary events.
`
`DR. REDDY’S LABS., INC. EX. 1019 PAGE 3
`
`

`

`1649
`
`Open forum
`
`individuals.
`if not varying amongst
`MDS patients to another,
`is in MOS,
`its consequences are
`Whatever
`the initial event
`very well appreciated. The affected cell develops a growth
`advantage over
`its neighbors eventually
`leading to mono-
`clonal hematopoiesis. Once the entire BM is descending from
`a single parent cell,
`the likelihood
`of additional mutations
`accumulating
`in one of
`its daughters is very great. Further,
`the evolved and additionally mutated cell will have a greater
`chance of survival and future proliferation
`in a monoclonal
`setting as compared to a polyclonal one because all
`the sur-
`rounding sister cells share in the same 'initial event' and thus
`are not very different
`from it. The expansion (and perhaps
`further mutations) of one such clone is probably responsible
`for the eventual evolution to acute leukemia. The key conse-
`quence of the stem cell defect
`in MDS then is monoclonality
`which predisposes these patients to frank malignancy.
`In fact,
`the definition
`of pre-leukemia
`should perhaps be rendered
`more objective by restricting the term to individuals in whom
`monodonality
`(in the absence of a substantial number of
`blasts) can be documented.
`
`What
`
`causes
`
`the clinical
`
`syndrome
`
`of MDS?
`
`The apparent paradox of variable cytopenias in the presence
`of generally
`cellular marrows
`in these patients could be
`accounted for by increased proliferation
`of hematopoietic
`cells being cancelled by an equally increased rate of intrame-
`dullary apoptosls. Thus, despite frantic activity in the marrow
`compartment,
`the peripheral blood would remain devoid of
`functional
`hematopoietic
`cells.
`In fact, our own studies
`directed at measuring the in vivo rates of proliferation and
`programmed cell death have demonstrated the above·hypoth-
`esis to be quite true. By infusing MDS patients with thymidine
`analogues iodo- and/or bromodeoxyuridine
`(lUdR, BrdU) as
`previously described,38.3Y we found that
`the total cell cycle
`time (Tc) in 120 MDS patients was a median of 35 h while
`the labeling index (L1)was 26.1%. Figure 2 shows a double-
`labeled slide in an MDS patient where large numbers of cells
`can be seen synthesizing DNA. Thus, MDS are clearly actively
`proliferative disorders with no dearth of S-phase cells in their
`bone marrows. Unfortunately,
`the rapid proliferation is coun-
`tered by rapid and excessive cell death.
`
`Double labeling for the two thymidine analogues iodo-
`Figure 2
`deoxyuridine (IUdRj (brown) and bromodeoxyuridine (BrdU) (blue)
`depicting the cells synthesizing DNA in the bone marrow biopsy of
`an MDS patient. Original magnification x 400.
`
`r I
`
`,erythroid, myeloid and megakaryocytic
`
`cells are all marked
`by the pathognomonic Philadelphia (Ph') chromosome."
`In
`CML, the initial
`'event' appears to be the Ph'
`translocation
`since the 8M resumes
`a polyclonal
`nature
`upon disappear-
`ance of this cytogenetic
`abnormality
`in response
`to interferon
`therapy."
`In MDS on the other hand, cytogenetic
`abnormali-
`5 or 7 appear
`to be
`epiphenomena or secondary events since they often involve
`a proportion of cells in a marrow which is otherwise mono-
`the first consequence of
`the
`is monoclonality
`and cytogenetic abnor-
`transforming event
`malities represent disease evolution while in CML,
`the Ph'
`• translocation may be the 'transforming event' since there is
`
`I ties such as those affecting chromosomes
`,clonal.l1,2h In MDS therefore,
`I
`I no evidence of monoclonal hematopoiesis upon eradication
`
`of the Ph' chromosome. The model of normal hematopoiesis
`proposed by Killman"
`is particularly
`relevant
`in this setting
`(Figure 1).
`
`Nature of the transforming
`in MDS
`
`event
`
`is obscure
`
`and ambiguous
`
`Since there are certain cytogenetic abnormalities which occur
`in MOS with a regular
`frequency,
`it
`is not unreasonable to
`look for genic abnormalities associated with the most com-
`monly involved areas of the affected chromosomes.
`In MDS,
`
`r the interstitial deletion involving 5q31.1 with its associated
`
`The 5q31.1
`loss of the [RF-l gene is one such abnormality."
`\ deletion is present in 30% MDS patients and 50% AML pati-
`f ents who developed leukemia secondary to a preceding mye-
`lodvsplasia."
`Located
`at - this
`site is the
`transcription
`enhancer/tumor suppressor JRF-l gene" which can also be
`functionally
`inactive
`due to accelerated
`'exon
`skipping'
`despite the presence of a. normal gene."
`Jt is also possible
`that exaggerated exon skipping- in the allele containing the
`normal JRF-l superimposed upon the deleted [RF-l allele
`accounts for the evolution of MDS to AML. Conversely, over-
`expression of
`IRF-2 can suppress the effects of
`IRF_l.31 The
`problem with implicating the IRF-l
`in the 'transforming event'
`isthat this deletion was detected in only 20-80% of the hema-
`topoietic cells in MOS patients correlating with the percentage
`of blasts> rather than the 100% of the cells expected on the
`basis of the monoclonal nature of the disease. ln other words,
`a genetic abnormality which only affects a proportion of the
`MDS cells instead of all
`the monoclonal
`cells by definition
`cannot be a candidate for the transforming event.
`Inappropriate expression of another gene,
`the esotropic
`virus integration site 1 gene (Evi-l) associated with retrovirally
`induced myeloid leukemias in mlce" has also been associ-
`ated with AML and MDS in humans.!" Aberrant expression of
`Evi-l conferred a non-responsiveness in murine BM progeni-
`tor cells to the effects of erythropoietin,
`an observation parti-
`cularly relevant to MDS since the erythroid cells appear to be
`universally most diseased in. these disorders. -Because abnor-
`malities of Evi-i are not found in all MDS patients, once again
`it is difficult
`to consider this gene as being somehow involved
`in the initial
`'transforming
`event'. Other molecular
`lesions
`which have been described include mutations of ras.> fms;"
`p5336 as well as deregulation of other as yet unidentified
`tumor suppressor genes."
`In summary therefore, MOS are clearly stem cell disorders
`although the nature of the initial event 'is unknown. Given that
`these complex. syndromes are highly heterogeneous in their
`clinical presentations,
`it
`is Ukely that
`the initial event(s) will
`parallel this heterogeneity· being different
`from one group of
`
`DR. REDDY’S LABS., INC. EX. 1019 PAGE 4
`
`

`

`I
`
`that
`is conceivable
`It
`cells.46-48
`apoptosis in hematopoietic
`of
`one or more of these cytokines are causing the stimulation
`early (034+ progenitor
`cells
`in the 8M while
`inducing I
`apoptosts in their maturing progeny.
`Indeed, we have demon-I
`strated higher
`than expected levels of all
`three c~tokin~s
`in
`MOS patients." What
`is the source of these cytoktnes! EIther ..
`I
`they are being produced by the transformed cells themselves
`they are being secreted by I
`as a mode of autostimulation
`or
`I
`the 'normal' monocytes/macrophages
`in the body's attempt
`to
`contain the expanding monoclonal population of transformed
`cells. Whatever
`the source,
`their effect
`IS detrimental at two
`levels since they end up stimulating the offending premature
`CD34+ blasts while destroying the maturing CD34- cells.
`
`\
`
`A paradigm shift
`
`I
`
`Figure 5 presents the new paradigm. A poorly defined initial
`event(s) leads to unrestrained growth of
`the abnormal
`cell
`by"
`eventually overwhelming
`the entire BM as demonstrated
`Presence of cytokines whose I
`studies of
`'monoclonality'.
`source also remains unexplained at the moment, confounds
`the .picture further by stimulating the early precursors and
`destroying the later forms most likely accounting for the clini-
`cal syndrome of pancytopenia and hypercellular
`bone mar-
`rows. The commonly encountered cytogenetic abnormalities
`since these are r
`events
`probably represent
`later evolutionary
`not usually present
`in every BM cell. When MOS patients .
`evolve to AML,
`there are distinct additional events,
`ie loss of
`\
`differentiation
`in the leukemic clone and therefore loss of
`t
`apoptosis,
`Implicit
`in the above is of course the loss of cvto-
`kine-induced effects and therefore an assumption of more
`autonomous growth by the leukemic clone. Treatment out-
`come of such secondary AML patients is distinctly inferior
`to
`primary AML probably because the cell
`involved is decidedly
`more primitive in the former
`(belonging to the 'feeder' pool)
`as compared with the latter
`(belonging to the 'committed'
`myeloid progenitor).
`
`Novel
`
`therapies
`
`resulting from the new paradigm
`
`By the time MOS patients present to their physicians with the
`full-blown clinical syndrome, several additional abnormalities
`have probably already followed the initial
`transforming event.
`Attempts to reverse each of
`the genic events may be some
`
`,,~~~
`timulate-
`..
`f Apoptosis
`IIIIIIJJII'~'
`
`Cytokines
`(? TNF-o:, TGF-I3,
`1L1I3/ICE)
`
`JS
`
`Monoclonal
`Hemopoiesis
`
`? IRF.1, Evi-1,
`ras, p53 etc
`
`Figure5
`
`The new paradigmfor MOS.
`
`1650
`
`Open forum
`
`In situ end labeling (lSEL)of fragmented DNA in MoS
`Figure3
`bone marrow biopsy showing high percentageof cells undergoing
`apoptosis. Original magnification x 400.
`-
`
`Apoptosis in' the 8M biopsies of MDS patients was meas-
`ured using the technique of in situ end labeling (ISEL)of frag-
`mented DNA40--44 and found to be very high as compared to
`normal or AML marrows.v'<':' Among 102 MDS patients stud-
`ied by ISEL, approximately
`half showed
`30-50% cells
`undergoing apoptosis in their biopsies. Figure 3 shows a typi-
`cal MDS BM biopsy. Double-labeling
`for proliferation
`(IUdR/BrdU) and apoptosis (ISEL) simultaneously
`showed
`large numbers of Scpbase cells to be apoptotic (Figure 4) rais-
`ing the question of 'antonymous' death in MDS.45 Thus, while
`the 8M in MDS patients is packed with proliferating cells, they
`are also Jn the process of concomitantly
`committing suicide.
`This may account
`for
`the paradox of pancytopenia despite
`hyperceJlularity of the marrow.
`
`Could dual-acting
`excessive
`cell-birth
`
`for the
`be responsible
`cytokines
`and cell-death
`in MDS bone marrows?
`
`A variety of cytokines such as tumor necrosis factor alpha
`(TNF-a.),transforming growth factor beta (TGF-J3)and lnterleu-
`kin 1 beta (IL-11l1 along with IL-11l converting enzyme (ICE)
`can have dual effects of stimulating proliferation and inducing
`
`Simultaneous presence of cell birth (brown) and cell
`Figure4
`death(blue)demonstratingthe phenomenaof antonymy in MoS bone
`marrow biopsy. Original magnification x 1000.
`
`DR. REDDY’S LABS., INC. EX. 1019 PAGE 5
`
`

`

`1651
`
`however may be a
`symptoms
`way in the future. The clinical
`result of
`the confounding
`actions
`of cytokines
`and therefore
`one could attempt
`to neutralize
`some of
`these effects with the
`expectation
`of clinical
`benefit.
`In the past,
`this approach
`of
`biotherapy
`in MDS has been restricted
`to the use of
`'viability
`(IL~3),granulocyte
`colonv-stlmu-
`factors'
`such as interleukin-3
`lating factor {G-CSFl or granulocyte-macrophage
`CSF (GM-
`CSF) and erythropoietin
`or
`their
`combinations.
`These trials
`have resulted
`in selective
`and variable
`benefits
`in a number
`of MDS patients. SO-52Unfortunately,
`the responses
`have been
`mostly
`transient
`and often the side-effects
`preclude
`prolonged
`trials.so-s2 Chemotherapeutic
`trials
`have been
`equally
`toxic
`and unimpressive
`and could
`only
`be attempted
`in patients
`with
`excess
`blasts. IS Combinations
`of growth
`factors
`and
`results." Obvi-
`chemotherapy
`have been tried with
`similar
`ously,
`these approaches may be refined
`further
`in the future
`cherno-
`when
`newer
`agents
`such
`as thrombopoietin
`and
`therapies
`such as topotecan
`are introduced
`into clinical
`trials
`to MDS ther-
`in MDS patients. We propose
`a novel approach
`apy based on the new paradigm. Why
`not
`try anticytokine
`therapy which would
`neutralize
`the noxious
`cytokines
`thereby
`producing
`a dual effect of ameliorating
`cytopenias
`(by sup-
`pressing apoptosis)
`and decrease the proliferation
`of
`the trans-
`formed
`done
`leading
`to resumption
`of polyclonality?
`What
`are the candidate
`agents
`in this area?
`TNF~a, TGF-,8 and
`First, a cascade
`of cytokines
`including
`IL-1{3 act
`(PAl -
`diacylglycerol
`the phosphatidic
`acid
`via
`Interruption
`of
`this
`(DAG)
`lipid
`signalling
`pathway.s4-s9
`such as oentoxifvlhne"
`second messenger
`system by drugs
`lisophvlline"
`and ciprofloxacin'" or
`could
`be tried. Second,
`specific
`inhibition
`of
`individual
`cytokines
`could
`be attempted
`receptor or IL~1,8 receptor
`by use of soluble TNF-o:
`antagonists
`in vivo. Finally,
`given to patients
`a combined
`'multi-modality'
`approach
`using viability
`factors and chemotherapy
`alternating
`with the anticytokine
`approach may be a far more valuable
`strategy than using anyone
`of
`the above
`alone.
`The idea is
`that a whole
`new area of clinical
`trials has been opened
`up
`by the construction
`of
`this new paradigm.
`Further
`therapeutic
`advances will occur only if we do not
`throw the baby out with
`the bath water and make sure that parallel
`detailed
`biological
`studies are conducted
`with the ·trial of every new agent(s)
`order
`to define the reasons for
`response and non-response.
`
`in
`
`Acknowledgements
`
`and Ms
`like to thank Ms Sandra Howery
`The authors would
`for expert
`secretarial
`and administrative
`Lakshmi Venugopal
`assistance. This work was supported
`by NCI grants CA60085
`and CA60086.
`
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