_ Journal
`Immunology
`
`G-CSF and GM-CSF in Neutropenia
`
`Hrishikesh M. Mehta, Michael Malandra and Seth J. Corey
`
`This information is current as
`of December 7, 2016.
`
`Jlmmunol 2015; 195:1341-1349; ;
`doi: 10.4049/jimmunol. 1500861
`http://www.j immunol, org/content/195/4/1341
`
`References
`
`This article cites 114 articles, 51 of which you can access for free at:
`http://www.j immunol, org/content/195/4/1341, full#ref-li st- 1
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`Copyright © 2015 by The American Association of
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`
`G-CSF and GM-CSF in Neutropenia
`Hrishikesh M. Mehta,* Michael Malandra, and Seth J. Corey*’’+
`
`G-CSF and GM-CSF are used widely to promote the
`production of granulocytes or APCs. The U.S. Food
`and Drug Administration approved G-CSF (filgrastim)
`for the treatment of congenital and acquired neutrope-
`nias and for mobilization of peripheral hematopoietic
`progenitor cells for stem cell transplantation. A polyeth-
`ylene glycol-modified form of G-CSF is approved for
`the treatment of neutropenias. Clinically significant
`neutropenia, rendering an individual immunocompro-
`mised, occurs when their number is < 150011~1. Current
`guidelines recommend their use when the risk for febrile
`neutropenia is >20%. GM-CSF (sargramostim) is ap-
`proved for neutropenia associated with stem cell trans-
`plantation. Because of its promotion of APC function,
`GM-CSF is being evaluated as an immunostimulatory
`adjuvant in a number of finical trials. More than 20
`million persons have benefited worldwide, and >$5 bil-
`lion in sales occur annually in the United States. The
`Journal of Immunology, 2015, 195: 1341-1349.
`
`F ew physician-scientists have made as great an impact on
`
`our understanding of hematology or improved the lives
`of patients, estimated at >20 million (1), with blood
`and cancer disorders as Don Metcalf, who died in December
`of 2014. Laboring at the Walter and Eliza Hall Institute in
`Melbourne, Australia throughout his 50-year career, Metcalf
`used semisolid medium and cell culture supernatants to
`discover hematopoietic progenitor cells (e.g., granulocyte/
`macrophage colonies) and their growth factors (e.g., G-CSF
`and GM-CSF). Increased purification of these and related
`growth factors, sometimes from hundreds of mice injected
`with endotoxin, led to the molecular characterization and
`cloning of G-CSF, GM-CSF, M-CSF, stem cell factor, and
`IL-3 in the 1980s (2).
`Hematopoiesis is a highly proliferative (N101° cells/d) dy-
`namic process driven by multiple hematopoietic growth
`factors/cytokines (Fig. 1A). The hematopoietic growth factors
`are multifimctional and are critical for proliferation, survival, and
`
`*Division of Hematology, Oncology and Stem Cell Transplantation, Department of
`Pediatrics, Ann and Robert H. Lurie Children’s Hospital of Chicago and Robert H.
`Lurie Comprehensive Cancer Center, Chicago, IL 60611; 1Division of Gastroenterol-
`ogy, Hepatology, and Nutrition, Department of Pediatrics, Ann and Robert H. Lm’ie
`Children’s Hospital of Chicago, Chicago, IL 60611; and *Dep~u-tment of Cell and
`Molecul~u" Biology, Northwestern University Feinberg School of Medicine, Chicago,
`IL 60611
`
`This work was supported by the National Institutes of Health, the American Society of
`Hematology, the Leukemia and Lymphoma Society, and the Cures Within Reach Foun-
`dation (all to S.J.C.).
`
`Address correspondence and reprint requests to Dr. Seth J. Corey at the current address:
`Virginia Commonwealth University Massey Cancer Center Sanger Hall, Room 12-024,
`
`www.jimmunol.org/cgi/doi/10.4049/jimmunol. 1500861
`
`differentiation of hematopoietic stem, progenitor, and precursor
`cells to a terminally differentiated, functional cell type. Colony-
`forming assays identified the ability of first crude supernatants,
`and then highly purified cytokines, to drive multi-lineage and
`single-lineage differentiation. After coculturing for 7-14 d, col-
`onies from mononudear cells obtained from the mouse spleen or
`bone marrow were measured in semisolid medium. Based on the
`characteristics of cells within a single colony, the lineage(s)
`governed by the cytokine was determined. Granulocytes make
`up the majority of WBCs in human circulation and play an
`integral role in innate and adaptive immunity. In granulopoi-
`esis, their production is mediated by a number of growth fac-
`tors, espedally G-CSF and GM-CSF (3, 4). Due to asymmetric
`division, some daughter cells of the hematopoietic stem cell
`(HSC) remain as HSCs, preventing the depletion of the stem
`cell pool (5). Multiparameter immtmophenotyping has trans-
`formed our ability to identify different cell types in hemato-
`poiesis. Murine HSCs are characterized as lin-scal +c-kit+, and
`human HSCs display CD34 in the absence of lineage markers.
`The differentiation pathway from HSCs to granulocytes is de-
`pendent on G-CSF and, less so, on GM-CSF. The HSC gives
`rise to a common myeloid progenitor and a common lymphoid
`progenitor cell (6). The common myeloid progenitor cells dif-
`ferentiate into myeloblasts, erythrocytes, and megakaryocytes via
`at least two intermediates: the granulocyte/monocyte progenitor
`cell and the erythrocyte/megakaryocyte progenitor cell. In the
`granulocytic series, myeloblasts (15-20 b~m) are the first rec-
`ognizable cells by their scant cytoplasm, absence of granules,
`and fine nudeus with nudeoli in the bone marrow dearly
`committed to differentiation to granulocytes. Myeloblasts dif-
`ferentiate into promyelocytes, which are larger (20 b~m) and
`begin to possess granules (Fig. 1B). Promyelocytes give rise to
`neutrophilic, basophilic, and eosinophilic precursor cells. Cell
`division continues through the promyelocyte stage. Fine spedfic
`granules containing inflammatory-related proteins appear dur-
`ing myelocyte maturation. For neutrophils, their size and
`nuclei become increasingly more condensed as the cells
`mature through myelocyte, metamyelocyte, band, and the ter-
`minally differentiated neutrophil (polymorphonudear and N15
`b~m). During episodes of stress, such as infection, band cells can
`
`1101 East Marshall Street, P.O. Box 980121, Richmond, VA 23298. E-mail address:
`coreylab@yahoo.com
`
`Abbreviations used in this article: AML, acute myeloid leukemia; ANC, absolute neu-
`trophil count; ASCO, American Society of Clinical Oncology; FDA, U.S. Food and
`Drug Administration; HSC, hematopoietic stem cell; IBD, inflammatmy bowel disease;
`IST, immunosuppressive therapy; MDS, myelodysplastic syndrome; NHL, non-Hodgkin’s
`lymphoma; PAP, pulmon~u’y alveol~u- proteinosis; SAA, severe aplastic anemia; SCN, severe
`congenital neutropenia.
`
`Copyright © 2015 by The American Association of hnmunologists, Inc. (X)22 1767/15/$25.(X)
`
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`
`1342
`
`TRANSLATING IMMUNOLOGY: G-CSF AND GM-CSF IN NEUTROPENIA
`
`A
`
`Multipotential Hematopoietic
`Stem Cell
`
`IL-1
`IL-3
`IL-6
`
`I GM-CSF
`.~SCF
`
`Common Myeloid Progenitor
`
`SCF
`TPO
`IL-3
`GM-CSF
`
`I SCF
`
`Epo
`IL-3
`GM-CSF
`
`GM-CSF
`G-CSF
`
`Megakaryocyte
`
`Myeloblast
`
`Early Lymphoid Progenitor
`
`FLT-3 Ligand / IL-2
`
`TNF-~1 IL-7
`TGF-61 IL-12
`SDF-1
`
`Common Lymphoid Progenitor
`
`B Lymphocyte
`
`T Lymphocyte
`
`Erythrocyte
`
`GM-CSF
`IL-3
`IL-4
`
`Platelets
`
`SCF
`G-CSF
`GM-CSF
`IL-3
`IL-6
`
`IL-3
`IL-5
`GM-CSF
`
`SCF
`M-CSF
`GM-CSF
`IL-3
`IL-6
`
`Basophil
`
`Neutrophil
`
`Eosinophil
`
`Monocyte
`
`More Differentiated
`
`Circulation
`
`B
`
`Less Differentiated
`
`Marrow Compartment
`
`Myeloblast
`
`Premyelocyte
`
`¯
`
`Neutrophilic
`Myelocyte
`
`¯
`
`Neutrophilic
`Metamyelocyte
`
`~_[ Neutrophilic
`Stab Cell (Band)
`
`¯
`
`Neutrophil
`
`[
`
`FIGURE 1. (A) Schematic diagram of hematopoiesis from the multipotential HSC to fully differentiated cell types. Principal cytoldnes that determine dif-
`ferentiation patterns are shown in red. (B) The stages of granulopoiesis from myeloblast to the mature granulocyte. During neutrophil maturation, which is driven
`primarily by G-CSF, granulocytic cells change shape, acquire primary and specific granules, and undergo nuclear condensation. Epo, erythropoietin; SCF, stem
`cell factor; SDF-1, stromal cell~lerived factor-l; TPO, thrombopoietin.
`
`be found in the peripheral blood and are used as a measure of
`inflammation. The above process is complex, dynamic, and
`orchestrated by multiple cytokines and their receptors, most
`notably G-CSF and GM-CSF.
`Following Ag stimulation or activation by cytokines, such
`as IL-1, IL-6, and TNF-o~, macrophages, T cells, endothelial
`cells, and fibroblasts produce and secrete G-CSF and GM-
`CSF. Of unknown significance, a variety of tumor cells also
`produce these paracrine growth factors. Glycoproteins with
`a molecular mass N 23 kDa, G-CSF and GM-CSF, are now
`produced through recombinant technology in either Escher-
`ichia coli or yeast. G-CSF induces the appearance of colonies
`containing only granulocytes, whereas GM-CSF gives colo-
`
`nies containing both granulocytes and macrophages. Gener-
`ation of G-CSF (Csf3)- and G-CSFR (Csf3r)-knockout mice
`confirmed that G-CSF critically drives granulopoiesis (7). The
`cognate receptor for G-CSF is a single-transmembrane receptor
`that homodimerizes upon G-CSF binding. Unlike G-CSF,
`GM-CSF functions via a two-receptor system involving a spe-
`cific c~-chain and a common [3-chain shared by IL-3 and IL-5
`(8). However, GM-CSF-knockout mice did not display a per-
`turbation in hematopoiesis (9, 10). Both G-CSF and GM-CSF
`signal through pathways involving JAK/STAT, SRC family
`kinases, PI3K/AKT, and Ras/ERKI/2. The receptor complexes
`are characterized by high-affinity (apparent KD N 100-500 pM)
`and low-density (50-1000 copies/cell). Interestingly, human
`
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`The Journal of Immunology
`
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`G-CSF is functionally active on murine myeloid cells, but
`human GM-CSF is not. The signaling spedfidty likdy involves
`nuances in the proximal postreceptor phosphoprotein networks
`and the distal gene regulatory networks. The molecular path-
`ways and their cross-interactions in determining lineage sped-
`fidty are critical to the development of more specific therapies.
`Cloning of human GM-CSF and its expression in bacterial
`and eukaryotic cells were achieved in 1985 at the Genetics
`Institute (11), and cloning of G-CSF and its expression in
`E. coli was achieved a year later at Amgen (12). Commerdal-
`ized by these biotechnology start-ups, G-CSF and GM-CSF
`revolutionized the treatment of patients with congenital or
`acquired neutropenias and those undergoing stem cell trans-
`plantation. Sidelined from the treatment of neutropenias by its
`toxicity profile, GM-CSF is now undergoing a renaissance as
`an immunomodulatory agent.
`G-CSF is approved by the U.S. Food and Drug Adminis-
`tration (FDA) for use to decrease the incidence of infection in
`patients with nonmyeloid malignancies receiving myelosup-
`pressive anticancer drugs associated with a significant incidence
`of severe neutropenia with fever; reduce the time to neutrophil
`recovery and the duration of fever following induction or
`consolidation chemotherapy treatment of patients with acute
`myeloid leukemia (AML); reduce the duration of neutropenia
`and febrile neutropenia in patients with nonmyeloid malig-
`nancies undergoing myeloablative chemotherapy followed by
`stem cell transplantation; mobilize hematopoietic progenitor
`cells into the peripheral blood for collection by leukapheresis;
`and reduce the incidence and duration of complications of
`severe neutropenia in symptomatic patients with congenital
`neutropenia, cyclic neutropenia, or idiopathic neutropenia.
`Forms of G-CSF available worldwide include filgrastim,
`pegfilgrastim, and lenograstim.
`GM-CSF is approved by the FDA to accelerate myeloid
`recovery in patients with non-Hodgkin’s lymphoma (NHL),
`acute lymphoblastic leukemia, and Hodgkin’s disease under-
`going autologous stem cell transplantation; following induction
`chemotherapy in older adult patients with AML to shorten
`time to neutrophil recovery and reduce the incidence of life-
`threatening infections; to accelerate myeloid recovery in
`patients undergoing allogeneic stem cell transplantation from
`HLA-matched related donors; for patients who have undergone
`allogeneic or autologous stem cdl transplantation in whom
`engraftment is delayed or failed; and to mobilize hemato-
`poietic progenitor cells into peripheral blood for collection
`by leukapheresis. Forms of GM-CSF available worldwide are
`sargramostim and molgramostim.
`The recommended dosage for G-CSF is 5 Ixglkgld and for
`GM-CSF is 250 Ixg/m2/d. Both drugs may be given s.c. or
`i.v., although randomized clinical trials demonstrate greater
`efficacy (i.e., decreased duration of neutropenia) without a dif-
`ference in toxidty for the s.c. route (13). For chemotherapy-
`induced neutropenia, G-CSF is administered until there are
`> 1000 neutrophils/Ixl. For congenital neutropenias, the goal is
`to maintain neutrophil counts N 75011xl. G-CSF is well toler-
`ated. Transient fever and bone pain are more commonly ob-
`served in those receiving GM-CSF. Pleural and/or pericardial
`effusions can also occur in those receiving GM-CSF. Long-term
`side effects of G-CSF administration, such as osteopenia, are
`being monitored in patients with severe congenital neutropenia
`(SCN). One concern is that G-CSF may accelerate the trans-
`
`formation of SCN to myelodysplastic syndromes (MDSs) or
`AML, associated with acquired mutations in G-CSFR.
`
`G- CSF and GM-CSF si~*aling l)athways and functional consequences"
`
`The receptors for both GM-CSF and G-CSF belong to the
`hematopoietin/cytokine receptor superfamily. G-CSFR acts as a
`homodimer, whereas GM-CSFR is a heterodimer with a shared
`[3-chain with the IL-3R and IL-5R complexes. G-CSFR is
`expressed primarily on neutrophils and bone marrow precursor
`cdls, which undergo proliferation and eventually differentiate
`into mature granulocytes. G-CSF binds to G-CSFR, resulting
`in its dimerization, with a stoichiometry of 2:2 and with a
`high affinity (KD = 500 pM) (14, 15). Among the activated
`downstream signal-transduction pathways are JAK/STAT, Src
`kinases, such as Lyn, Ras/ERK, and PI3K (16). The cytoplas-
`mic domain of G-CSFR possesses four tyrosine residues (Y704,
`Y729, Y744, Y764) serving as phospho-acceptor sites (17, 18).
`Src homology 2-containing proteins STAT5 and STAT3 bind
`to Y704 and Gab2 to Y764. Grb2 couples to both Gab2 and to
`SOS, permitting signaling diversification, such as Ras/ERK,
`PI3K/Akt, and Shp2 (19, 20). An alternatively spliced iso-
`form of G-CSFR elidts activation of a JAK/SHP2 pathway
`(15). The predse physiological roles of protein kinases and
`their downstream events in G-CSF-induced signaling remain
`undear, although some dues are beginning to emerge (21, 22).
`GM-CSF binds to the o~-chain of the GM-CSFR with a
`low affinity (KD = 0.2-100 nM), but a higher affinity (K> =
`100 pM) occurs in the presence of both subunits. GM-CSF
`signaling involves the formation of dodecameric supercomplex
`that is required for JAK activation (23). In addition to the JAK/
`STAT pathway, GM-CSF activates the ERKI/2, PI3K/Akt,
`and IKB/NF-KB pathways. Although the o~-chain is primarily
`considered a ligand-recognition unit, it interacts with Lyn,
`resulting in JAK-independent Akt activation of the survival
`pathway (24). Thus, differences in receptor expression patterns
`and known and unknown nuances in signaling pathway circuits
`account for the functional differences between G-CSF and
`GM-CSF.
`G-CSF and GM-CSF are pleiotropic growth factors, with
`overlapping functions. GM-CSF also shares properties with
`M-CSF on monocyte function (25). Both GM-CSF and G-CSF
`increase chemotaxis and migration of neutrophils, but response
`kinetics may differ. GM-CSF may be considered to be more
`proinflammatory than G-CSF. GM-CSF increases cytotoxic
`killing of Candida albicans, surface expression of Fc- and
`complement-mediated cell-binding (FcyR1, CR-1, CR-3),
`and adhesion receptor (14). Yet, both cytokines promote
`neutrophil phagocytosis (26). More extensive reviews on
`G-CSF and GM-CSF function in neutrophils may be found
`(27, 28).
`
`Acquired and congenital neutrot)enia
`
`An absolute neutrophil count (ANC) < 150011xl is defined as
`neutropenia, which is graded on the severity of decreased
`ANC (Table I). Causes for neutropenia may be congenital
`or, more commonly, acquired. Neutropenia may be asymp-
`tomatic until an infection occurs. Benign neutropenia exists,
`and the individuals are not at risk for serious infection.
`However, onset of fever with neutropenia, termed febrile
`neutropenia, commonly occurs as a potentially life-threatening
`complication of chemotherapy and involves considerable cost as
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`TRANSLATING IMMUNOLOGY: G-CSF AND GM-CSF IN NEUTROPENIA
`
`Table 1. Correlation of neutropenia with absolute neutrophil count
`
`Neutropenia Grade
`
`Absolute Neutrophil Count
`
`Grade 1
`Grade 2
`Grade 3
`Grade 4
`
`->1.5 to <2 )< 109/ml
`->1 to <1.5 )< 109/ml
`->0.5 to <1 × 109/ml
`<0.5 × 109/ml
`
`a result of treatment with i.v. antibiotics and prolonged hospi-
`talization. In addition, febrile neutropenia prevents continuation
`of chemotherapy until recovery from it occurs. According to the
`Norton-Simon hypothesis (29), the efficacy of chemotherapy is
`reduced if stopped midway. A pause in treatment allows re-
`covery of the cancer cells and facilitates the emergence of chemo-
`resistant dones (29-31). Neutropenia also occurs secondary to
`bone marrow infiltration with leukemic or myelodysplastic cells.
`Neutropenia results from a growing list of germline mu-
`tations in genes, such as ELANE, HAX1, GFI1, G6PC3, WAS,
`and CSF3R (32). Soon after birth, children with SCN develop
`a grade 4 neutropenia. SCN is a lifetime condition resulting
`from increased apoptosis of granulocytic progenitors in the
`marrow. As a result of the severity and chronic nature of
`SCN, individuals are prone to recurrent infections, especially
`from the endogenous flora in the gut, mouth, and skin. Most
`cases of SCN are due to de novo mutations. Transmission
`may be autosomal dominant, recessive, or X-linked. The most
`common mutation involves ELANE and is autosomal domi-
`nant (33, 34). Mutations in ELANE encode the neutrophil
`elastase, a serine protease. ELANE is expressed during gran-
`ulopoiesis, maximally at the promyelocyte stage. It is hy-
`pothesized that mutations in ELANE cause neutropenia via
`improper folding of the protein that triggers the unfolded
`protein response. Unfolded protein response-generated stress
`drives apoptosis due to an overload of unfolded protein, and
`an arrest in differentiation at the promyelocyte stage is ob-
`served. Fascinatingly, ELANE mutations are also associated
`with cyclic neutropenia. Cyclic neutropenia is characterized
`by granulocyte nadirs < 20011_tl occurring every 21 d.
`Patients with SCN are always at risk for life-threatening
`infections. Early phase 1 dinical trials held in 1989 (35, 36)
`evaluated G-CSF therapy for SCN and cyclic neutropenia.
`Both trials demonstrated a -> 10-fold increase in neutrophil
`counts, reducing the severity of neutropenia from grade 4 to
`grade 1 to normal counts. A reduction in the days of cyclic
`neutropenia from 21 to 14 d was observed, and a consistent
`increase in ANC was observed in SCN. In 1990, two studies
`explored the benefit of G-CSF versus GM-CSF in treating
`congenital neutropenia. Gray collie dogs with cyclic neu-
`tropenia due to mutations in the endocytosis gene AP3BI
`(37) were studied with three cytokines: G-CSF, GM-CSF,
`and IL-3. GM-CSF and G-CSF showed an expansion of
`neutrophil counts, but only G-CSF prevented the cycling of
`hematopoiesis (10). Similar to the dog study, G-CSF therapy
`increased ANC, whereas GM-CSF therapy increased eosino-
`phil counts but not neutrophil counts (38). Following the
`beneficial effects of G-CSF in the above phase 112 studies,
`a phase 3 dinical trial was performed in 1993 (39). Patients
`with SCN, cyclic neutropenia, and idiopathic neutropenia
`(n = 123) were induded in the study. Patients were randomly
`treated immediately or after a 4-mo observation period. Al-
`most all of the patients (1081120) receiving G-CSF therapy
`
`displayed a restoration of ANC from grade 4 to normal levels.
`The increase in ANC resulted from increased production of
`neutrophils in bone marrow. Infection-related incidents were
`reduced by N50% (p < 0.05), and antibiotic use was reduced
`by 70%.
`One particular form of inherited neutropenia is WHIM
`(warts, hypogammaglobulinemia, infections, and myeloka-
`thexis) syndrome (40). Myelokathexis refers to a build-up of
`mature neutrophils in the bone marrow. Mutations in CXCR4
`result in the syndrome (41). CXCR4 and its ligand SDF-1
`mediate the retention of neutrophils. G-CSF administration
`leads to upregulation of SDF-1 and the subsequent release of
`neutrophils into the peripheral circulation (42). A recently
`published phase 1 study demonstrated the safety and efficacy
`of low-dose plerixafor, a CXCR4 antagonist (43). One widely
`used indication for G-CSF is to mobilize and harvest hema-
`topoietic progenitor cells into the periphery for stem cell
`transplantation (44), and the concomitant use of plerixafor
`enhances the mobilization (45).
`
`Neutropenia associated with aplastic anemia
`
`Severe aplastic anemia (SAA) is a disease in which stem cells
`residing in the bone marrow are damaged, leading to a defi-
`ciency in all hematopoietic cell lines. SAA has a high mortality,
`but the 5-y mortality is reduced to < 10% with matched sibling
`stem cell transplantation or to 30% with immunosuppressive
`therapy (IST) (46). IST includes antithymocyte globulin, cy-
`closporine, and glucocorticoids. The addition of G-CSF to IST
`was studied in a number of randomized studies. It was shown
`that G-CSF reduces the number of infectious complications
`and hospital days compared with standard therapy alone;
`however, its addition did not affect overall survival rates (47,
`48). Although treatment with G-CSF or GM-CSF results in
`a neutrophil response, a sustained trilineage response was un-
`common when used alone or in combination with other he-
`matopoietic growth factors (49, 50). The response to G-CSF
`may have prognostic value. Patients treated with IST plus
`G-CSF who did not achieve a WBC count -> 500011_tl had a
`low probability of response and high mortality (51-53). Sim-
`ilarly, GM-CSF was studied as a potential adjunct to IST with
`similar results (48). These finding suggest that G-CSF and
`GM-CSF may be useful adjuncts to standard IST for SAA.
`
`Neutropenia associated with chemotherapy for solid tumors
`
`In 1991, the FDA approved the use of recombinant human
`G-CSF (filgrastim) to treat cancer patients undergoing mye-
`lotoxic chemotherapy. Multiple factors affect the severity of
`neutropenia, with the most important being the type and
`severity of chemotherapy dosage and the underlying disease
`(54, 55). In 1994, the American Sodery of Clinical Oncology
`(ASCO) recommended primary prophylaxis with G-CSF or
`GM-CSF for the expected incidence of neutropenia -> 40%
`(56). The purpose of the guidelines was to reduce the inci-
`dence and length of neutropenia and, thus, the time of hos-
`pitalization, which would reduce costs significantly. Three
`prospective, randomized, placebo-controlled trials formed the
`basis of the recommendations. The first phase 3 trial tested the
`applicability of G-CSF as an adjunct to chemotherapy in
`patients treated for small cell lung cancer with cyclophospha-
`mide, doxorubicin, and etoposide (57). A major outcome of
`the study was the significant reduction by at least one episode
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`The Journal of Immunology
`
`1345
`
`of febrile neutropenia in 77% of those treated with G-CSF
`versus 40% in the placebo group. A reduction in the median
`duration of grade 4 neutropenia was observed in all cycles of
`chemotherapy (1 d in the G-CSF group versus 6 d in the
`placebo group). From a cost-benefit perspective, the data
`translated into a reduction in the 50% incidence of infection,
`antibiotic treatment, and days of hospitalization with G-CSF
`treatment versus placebo. A similar study performed in Europe
`in patients with small cell lung cancer also found that pro-
`phylactic G-CSF treatment reduced the incidence of febrile
`neutropenia (53% in placebo group versus 26% in G-CSF
`group) (58). A reduction in chemotherapy dose by 15% was
`indicated in 61% of the placebo group versus 29% of the G-CSF
`group. A gap -> 2 d in the chemotherapy treatment group
`was observed for 47% of patients in the placebo group and
`29% of the patients in the G-CSF group. The third trial in-
`vestigated G-CSF therapy in NHL treated with vincristine,
`doxorubicin, prednisolone, etoposide, cydophosphamide, and
`bleomycin (59). The incidence of neutropenia was reduced for
`the G-CSF group (23%) versus the placebo group (44%), with
`fewer ddays and shorter duration of treatment in the G-CSF-
`treated group. In comparison, GM-CSF trials provided less
`convincing data. In a trial for cydophosphamide, vincristine,
`procarbazine, bleomycin, prednisolone, doxorubicin, and
`mesna administered as therapy for NHL, the use of mol-
`gramostim (GM-CSF) resulted in faster recovery from neu-
`tropenia and reduced hospitalization, but the benefit was
`limited to only 72% of the patients that could tolerate GM-
`CSF (60). Another trial with small cell lung cancer did not
`show any significant effect with molgramostim treatment (61).
`The development of better chemotherapeutic regimens that
`were less myelotoxic provided more cost-effective options
`compared with CSF therapy. In many cases, the incidence of
`neutropenia was reduced to -< 10%. However, the advantage
`of CSF therapy in both increasing the intensity and mainte-
`nance of dose versus the cost of the growth factors was actively
`debated. In 2003, a large randomized study showed the
`benefit of G-CSF therapy with dose-dense chemotherapy
`(cyclophosphamide, paditaxel, and doxorubicin) in patients
`with node-positive breast cancer (62). Significantly improved
`disease-free survival (risk ratio = 0.74; p = 0.01) and overall
`survival (risk ratio = 0.69, p = 0.013) were observed in
`patients receiving G-CSF. Fewer patients reported grade 4
`neutropenia in the G-CSF group (6%) compared with the
`non-G-CSF group (33%). In 2004, two additional studies
`with old (60-75 y) and young (<60 y) NHL patients ob-
`served a reduction in chemotherapy regimens from 3 to 2 wk
`combined with an improvement in the rate of progressive
`disease and overall survival (63, 64). In 2005, two large trials
`supported the use of G-CSF in redudng the incidence of fever
`and neutropenia and suggested its use with the first cyde of
`chemotherapy (65, 66). The first study compared the effect of
`antibiotics versus antibiotics ÷ G-CSF in small cell lung
`cancer patients undergoing cyclophosphamide, doxorubicin,
`and etoposide treatment. A significant reduction in the inci-
`dence of febrile neutropenia was observed for the antibiotics +
`G-CSF group (10%) compared with the antibiotics-only
`group (24%) (66). The second study investigated the effect
`of pegfilgrastim in breast cancer patients treated with docetaxel
`(65). Approved in 2001, pegfilgrastim was developed to im-
`prove the renal clearance rate (67), and a single dose provided
`
`similar or greater improvement in the ANC after chemotherapy
`compared with daily doses of filgrastim (68). The randomized,
`placebo-controlled trial conducted with 928 patients demon-
`strated a lower incidence of febrile neutropenia in patients
`receiving pegfilgrastim (1%) compared with placebo (17%).
`Hospitalization also was reduced in the pegfilgrastim group
`(1%) compared with the placebo group (14%). In 2005 and
`2006, the National Comprehensive Cancer Network (http://
`www.nccn.org) and ASCO changed the risk threshold for
`contracting neutropenia from 40 to 20% to justify the use
`of myeloid growth factors as an adjuvant to chemotherapy
`in treating neutropenia (69). The use of myeloid growth
`factors, their cost effectiveness, and the duration of their use
`during chemotherapy remain of great interest to dinical oncol-
`ogists. A randomized phase 3 study with a noninferioriry design
`demonstrated the efficacy of G-CSF prophylaxis against febrile
`neutropenia in women with breast cancer for the entirety of
`their myelosuppressive treatment (70). Current guidelines
`from ASCO, the National Comprehensive Cancer Network,
`and the European Organization for Research and Treatment
`of Cancer recommend the use of myeloid growth factors when
`the risk for febrile neutropenia is ->20% (71, 72).
`
`Neutropenia associated with leukemia
`
`Neutropenia in patients with leukemia results from both the
`underlying disease and aggressive chemotherapy. The ASCO
`guidelines developed in 1994, like for solid tumors, considered
`data obtained from three large randomized trials. Unlike the
`solid tumor trials, two of the three trials used GM-CSF versus
`G-CSF. The two GM-CSF trials reported conflicting findings,
`with some statistical significance in the recovery of ANC but no
`significant reduction in hospitalization or the inddence of serious
`infections (73, 74). The G-CSF trial showed a recovery in ANC,
`reduction in days of neutropenia, and a trend toward better
`recovery rates. However, like the GM-CSF trials, no improve-
`ment in days of hospitalization or usage of antibiotics was ob-
`served (75). Thus, a benefidal response by the growth factors
`was not observed in leukemia. However at the time of ASCO’s
`2000 guidelines, newer placebo-controlled trials demonstrated
`a reduction in neutrophil recovery time from 6 to 2 d and re-
`duced hospitalization times in the setting of induction chemo-
`theraW (76). The 2000 ASCO guidelines also identified a
`potential benefit for growth factor therapy in consolidation
`chemotherapy. The 2006 update did not introduce any signif-
`icant changes and recommended the application of CSF therapy
`postinduction and consolidation therapy (69).
`Unlike chemotherapy-induced neutropenia, congenital neu-
`tropenia patients experience neutropenia for life and require
`long-term treatment with G-CSF. The long-term effects of
`G-CSF therapy have become important in the management of
`congenital neutropenia. Patients receiving G-CSF therapy for as
`long as 8 y were evaluated for safety and efficacy (77). Neu-
`trophil counts were maintained without exhaustion of myelo-
`poiesis. A significant improvement in the quality of life was
`achieved by the reduction in antibiotic treatment and hospi-
`talization time, allowing for normal growth, development, and
`partidpation in normal daily activities. The SCN international
`registry was formed in 1994 to further assess the progress of
`SCN patients being treated with G-CSF. A 10-y report that
`followed patients with SCN (n = 526) who were being treated
`with G-CSF was released in 2006 (78). Consistent with pre-
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1096-0006
`
`

`
`1346
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`TRANSLATING IMMUNOLOGY: G-CSF AND GM-CSF IN NEUTROPENIA
`
`vious reports, an increase in ANC was observed in majority of
`the patients with an overall improvement in quality of life.
`Leukemia transformation is significantly higher in SCN
`patients, and the SCN international registry reported that 21%
`of patients with SCN developed leukemia while being treated
`with G-CSF. Although leukemic transformation has been
`reported in SCN patients before the development of G-CSF
`therapy (79), the precise role of G-CSF therapy in leukemic
`transformation remains unknown. Almost all SCN patients
`undergo G-CSF therapy; thus, it is difficult to assess leukemic
`transformation in the absence of G-CSF treatment. However,
`patients w