Management of Neutropenia

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Management of Neutropenia G-CSF vs Granulocyte
Transfusion
Sung-Soo Yoon
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Kinetics of neutrophil production
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Risk Factors for Serious Infection

• Neutropenia
• -Neutrophil lt 1 x 109/L, lt0.5 x 109/L
• -Fungal infection ? at neutrophil lt 0.2x109/L
• Duration of neutropenia
• Rate of fall of neutrophil count

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G-CSF

• 1st line of defense against infection PMN,
  macrophage, NK cells, cytotoxic lymphocyte
• Immunomodulatory effects of G-CSF
• Increase leukocyte count
• Upregulate phagocyte function during neutropenia
• Increase leukocyte count for apheresis purposes

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G-CSF

• Production monocyte/macrophage, fibroblast,
  endothelial cells
• G-CSF gene chromosome 17 q21-22
• G-CSF receptor gene chromosome 1 p35-p34.3
• Structure 4 helices connected by amino acid
  loops, O-glycosylated, 20 kD

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Available G-CSFs

• ? Filgrastim
• source E.coli
• structure nonglycosylated
• ? Lenograstim
• source CHO cell (Chinese Hamster Ovary cell)
  
• structure glycosylated

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Structure of Filgrastim
Pro
Gln
Ala
Leu
His
Arg
Arg
Leu
Val
Tyr
Ser
Val
Leu
Glu
Phe
Ser
Gln
Leu
His
Ser
170
160
Ala
10
0
Val
Pro
Leu
Ser
Ser
Ala
Pro
Gly
Leu
Pro
Thr
Met
Leu
Gln
Val
Ser
SH
20
150
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Gly
Phe
Arg
Glu
Leu
Cys
Lys
Leu
Leu
Asp
Lys
Val
Gln
Gln
Leu
Ala
Ala
Gly
Gly
Gln
Ile
Cys
Leu
Lys
Glu
Gly
Ala
Thr
30
S
Ala
Tyr
S
60
50
40
Arg
Lys
Cys
Ser
Ser
Leu
Pro
Ile
Ser
Pro
Ala
Thr
Pro
Gly
Leu
Ser
His
Gly
Pro
His
Cys
Leu
Leu
Val
Leu
Glu
Glu
Leu
Arg
Gln
S
Gln
Ala
Phe
Leu
S
70
Ala
Gln
80
Leu
Ser
Ala
Ser
Ile
Gly
Glu
Leu
Ala
Gln
Leu
Leu
Gln
Leu
Phe
Leu
Gly
Ser
His
Leu
Gln
Ser
Leu
Cys
Gly
Pro
Gly
Tyr
Ala
Glu
90
140
Phe
Leu
Gly
Ala
100
Leu
Asp
Leu
Thr
Ala
Phe
Asp
Ala
Asp
Leu
Gln
Thr
Pro
Val
Pro
Thr
110
Met
Thr
Ala
130
120
Ile
Gly
Trp
Gln
Leu
Ala
Thr
Pro
Gln
Pro
Ala
Met
Gly
Leu
Glu
Glu
Met
Gln
Gln
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Structure of Lenograstim
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Role of G-CSF

• Major target cells neutrophil precursors and
  mature neutrophils
• G-CSF knock-out mouse
• - mice rendered G-CSF deficient by targeted
  disruption of G-CSF gene in embryonic stem cells
• - 50 reduction of granulocyte precursor cells
  in the bone marrow
• - markedly impaired ability to control infection

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G-CSF for CD34 PBSC

• Presence of hematopoietic cells in peripheral
  blood after cytotoxic chemotherapy
• Similar response in normal persons after HGFs
• Benefits of PBSC
• Relative ease of procurement
• More rapid restoration of blood counts
• Recovery of cells in pts with prior chemo or
  radiotherapy
• Recovery of cells in pts with tumors involving
  bone marrow

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G-CSF for Chemotherapy-Induced Neutropenia

• Proven efficacy in randomized trial of SCLC pts
  Method
• SCLC with cytoxan, doxorubicin, etoposide
  chemotherapy
• G-CSF begin on day 4 through day 17 vs placebo
• 211 pts enrolled toxicity assay in 207, efficacy
  in 199 pts
• Results G-CSF vs Placebo
• at least one episode of fever 40 vs 77
• ANClt0.5 x 109/l 1 day vs 6
  days
• Antibiotic treatment, hospitalization, confirmed
  infection decrease by 50 in G-CSF group
• (NEJM 1991, Eur J Cancer 1993)

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G-CSF after Hematopoietic Stem Cell
Transplantation

• Randomized trial of breast ca pts
• Method
• Arm A, 5ug/kg/day start on day 0, arm B,
  5ug/kg/day start on day 5, Arm C, no G-CSF
• Results (G-CSF vs Placebo)
• Significantly earlier neutrophil engraftment in
  G-CSF arm
• Cf. Other factors to be considered
• - conditioning regimen
• - PBSC dose transfused
• - Patients condition
• (Bone Marrow Transplant 2002, Rev Invest Clin
  2002)

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G-CSF in Immunocompetent Patient

• Potential indications
• Sepsis prophylaxis time point for risk of
  infection known or anticipated (surgery, trauma,
  burn, local infection)
• - data available from several studies in pts
  with pneumonia, HIV infection, surgery, IDDM
  foot infections
• Esophagectomy for esophageal cancer pt
• - 155 pts randomized to phase III trial
• - perioperative G-CSF or placebo 2 days before
  surgery for a total of 10 days
• - no difference in morbidity, infection,
  mortality

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G-CSF in Immunocompetent Patient
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2000 ASCO Guidelines for CSFs
1.Primary prophylactic CSF administration -
Reduce the incidence of febrile neutropenia by
50 if FN incidence 40 - Consider CSF if
preexisting neutropenia due to ds, extensive
prior chemo, previous irradiation to pelvis or
other large marrow areas, history of recurrent
FN, certain conditions (poor performance status,
more advanced ca, decreased immune function, open
wounds, already-active tissue infections)
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2000 ASCO Guidelines for CSFs
2.Secondary prophylactic CSF administration -
except curable tumors (e.g., germ cell tumor),
consider dose reduction 3. Guidelines for CSF
therapy A. Afebrile patient not routinely
recommended B. Febrile patient not for
uncomplicated fever and neutropenia (fever 10
days, free of pneumonia, cellulitis, abscess,
sinusitis, hypotension, multiorgan dysfunction,
invasive fungal infection, uncontrolled
malignancies
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2000 ASCO Guidelines for CSFs
4.To increase chemotherapy dose-intensity - not
justified outside of a clinical trial 3.
Guidelines for CSF therapy A. Afebrile patient
not routinely recommended B. Febrile patient
not for uncomplicated fever and neutropenia
(fever 10 days, free of pneumonia, cellulitis,
abscess, sinusitis, hypotension, multiorgan
dysfunction, invasive fungal infection,
uncontrolled malignancies
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2000 ASCO Guidelines for CSFs
5.Adjunct to progenitor cell transplantation -
higher dose?greater CD34 cells in PBSC
product 6. Guidelines for acute leukemia,
MDS A. AML consider cost-benefit B. MDS
subset of pts with recurrent infection C. ALL
(newly added) - recommended after
chemotherapy - can be given with
corticosteroid/antimetabolite D. Leukemia in
relapse (newly added) - not enough evidence
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2000 ASCO Guidelines for CSFs
7.Concurrent chemotherapy/irradiation - avoid
CSFs, particulary involving the mediastinum 8.
Pediatric population - same as adult, dose to be
defined 9. Dose - 5 ug/kg/d for G-CSF,
250ug/m2/d for GM-CSF - for mobilization
10ug/kd/d
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G-CSF Toxicity

• Bone, musculoskeletal pain 20-30
• Anemia, thrombocytopenia, injection side
  reactions
• Sweets syndrome 2
• Spleen enlargements, respiratory disturbances
• Risk of malignant transformation?
• - tumor progression in animal study (Int J
  Cancer 1999)
• Potential long-term risk unknown

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Sweets syndrome
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Potential Long-Term Risk of G-CSF
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New Formulations PEG-G-CSF

• Adding polyethylene glycol (PEG) molecule to
  N-terminus of G-CSF molecule (pegylation)SD/01
• Plasma clearance? , plasma t1/2 ?
• Serum concentration of SD/01 increased for
  several days after a single injection
• Not associated with significant toxicity

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Neutropenia and Hematopoieitic Stem Cell
Transplantation

• Lack of responsive hemopoietic precursors

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History of Granulocyte Transfusion

• Granulocyte from CML pts
• 70s, survival? in G(-) sepsis with
  granulocytopenia 10 days
• 80-90s, disuse of granulocyte transfusion
• Inadequate dose
• Rapid apoptosis of granulocytes

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History of Granulocyte Transfusion

• Importance of granulocyte dose
• - retrospective analysis of 7 controlled trials
• - animal sepsis model, miningitis model
• Traditional dose lt20-30 x 109 cells (1/2 of
  daily marrow production in a normal, non-infected
  adult
• Benefit if a daily granulocyte dose gt 10 x1010
  cells
• Donor treatment
• - corticosteroid 2-fold PMN ? in peripheral
  blood
• - G-CSF 7-fold PMN ? in peripheral blood

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G-CSF to Stimulate Granulocyte Donors

• G-CSF ? mean granulocyte count up to 82 x 109
  cells (2-4 fold higher than corticosteroid
  priming only)
• Highest yields by administering both G-CSF/
  dexamethasone 12 h before collection
• Normal granulocyte function maintained
• Antifungal activity (e.g.,Aspergillus, Rhizopus)
  may be enhanced
• Prolonged intravascular survival when reinfused
• Localize to inflammatory sites

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Hematologic Effects in Recipients

• Large post-transfusion neutrophil increment with
  transfusion from G-CSF stimulated donors
• Sustained and maintained above baseline for 24h
  or more

• Study Cell Dose Cell Increment
• Hester 41 x 109 cells 0.6 x 103 /ul (J Clin
  Apheresis 1995)
• Adkins 51 x 109 cells 1 x 103 /ul
  (Transfusion 1997)
• Price 82 x 109 cells 2.6 x 103 /ul (Blood 2000)

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Factors for Prolonged Survival of PMN

• Shift of relatively young cells into the donors
  circulation from the marrow
• Antiapoptotic effect of G-CSF
• Normal migration of transfused cells to
  inflammatory sites

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Neutrophil Storage and Function

• Chemotaxis activity preserved for 24 hrs at room
  temp
• NADPH oxidase activity maintained for 48 hrs
• Slight ? of IL-1b, IL-8/ IL-6, TNF not changed
• Storage for 24h 48h at 22 10 C
• - respiratory burst activity greatest at 10 C,
  relatively preserved at 22 C
• - Bactericidal (vs Staphylococcus aureus),
  fungicidal (vs Aspergillus fumigatus, Rhizopus
  arrhizus, Candida albicans) activity maintained
  during storage for 48 h at 10 C

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Relationship btw PMN dose and ANC increment 1 h
post-transfusion
Price, Blood 2000
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Effect of serial neutrophil transfusions on the
ANC
Price, Blood 2000
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Requirements
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Granulocyte Collection/Storage

• Collection
• - Centrifugal leukapheresis by processing 7-10 L
  of blood over 3-4 hours
• - Hydroxyethyl starch to reduce RBC
  contamination
• - yield 2-3 x 1010/leukapheresis, 200-400 ml
  plasma, 10-30 ml of RBCs, 1-6 x 1011 platelets
• Storage
• - up to 24 hours at room temperature
• - preferable to transfuse within 8 hours of
  collection
• - effective storage possible for up to 48 hrs at
  10C

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Adverse Effects in Recipients

• Transfusion reaction

• - 10-50 incidence, usually mild
• more severe reaction in 1-5, usually pulmonary
• (no correlation with neutrophil dose)
• - serious potential interaction with amphotericin
  B 8 hours between amphotericin B and
  granulocyte transfusion (NEJM 19813041185-1189)
  
• transfusion associated GVHD prevented by
  1,500-3,000 cGy irradiation

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Adverse Effects in Recipients

• Alloimmunization

• Against HLA class I, granulocyte-specific
  antigens, especially after repeated transfusions
• Definitive information scare
• Rapid alloimmunization probably not possible in
  severely immunosuppressed pts
• Difficulty distinguishing reactions involving
  other components (RBC, platelet)
• Current standard check for antibodies for ABO
  antigens, leukoagglutination prior to transfusion

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Clinical Efficacy

• Problems
• - Most studies uncontrolled, small
  heteregeneous populations, different treatment
  approaches
• - 32 papers 62 of 206 pts with bacterial
  sepsis benefited, 71 of 63 pts with invasive
  fungal infections did not
• Efficacy reported if
• - Higher neutrophil doses
• - leucocyte compatibility

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Summary of 32 reports of infections treated with
granulocyte transfusion therapy in neutropenic
patients
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Potential Clinical Indications

• Severely neutropenic pts with bacterial
  infections
• Invasive fungal infections
• Prophylactic use in hematologic malignancies and
  transplantation setting
• Infected pts with severe neutrophil dysfunction
  (chronic granulomatous disease, leukocyte
  adhesion deficiency)
• Neonatal bacterial sepsis

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Neonatal Sepsis
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Preliminary Guidelines

• At least 96 hours after the onset of clinical
  signs and symptoms of infection
• Continue granulocyte transfusion for at least 7
  days
• Prophylactic granulocyte transfusion?

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Clinical efficacy of granulocyte transfusion in
severe neutropenic infections

• Jin-Soo Kim1, Sang-Il Kim1, Sook-Ryun Park1, Ji
  Yeon Baek1, In Sil Choi1, Sung-Soo Yoon1, Jong
  Suk Lee1, Seonyang Park 1 and Byoung Kook Kim 1
• 1. Seoul National University Hospital, Seoul,
  Republic of Korea

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Objectives

• To evaluate the safety and efficacy of
  granulocyte transfusion
• To identify favorable prognostic factor for
  granulocyte transfusion

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Materials and Methods

• May 1998 July 2003
• Granulocyte collection
• Healthy and ABO matched donor
• Mostly CS 3000 blood cell separator
• Sedimenting agent Pentaspan 500ml
• Target cell dose gt 1.0x1010 of granulocyte
• Granulocyte transfusion
• Transfuse over 1 hr, within 2hr of pheresis
• Protocol for granulocyte transfusion
• Donor preparations 12hr prepheresis
• G-CSF 300ug sc
• Dexamethasone 8mg po
• Recipient premedications
• Pheniramine 10mg iv
• Hydrocortisone 100mg iv two times

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Patient Selection

• Clinically or microbiologically documented severe
  infections
• Prolonged neutropenia (ANClt500) for at least 1wk
• Deterioration of clinical condition or no
  response of broad-spectrum antibiotics and/or
  antifungal agent
• No response to G-CSF therapy
• Informed consent

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Response Evaluation

• Definition of infection control
• Stabilization of vital sign(esp, control of
  fever)
• Improvement of CRP
• Negative conversion of bacteremia
• Improvement of pulmonary infiltrates in chest
  X-rays

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Patients Characteristics
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Result
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Toxicities
3 peri-transfusion mortality 1 massive
hemoptysis 2 progressive sepsis
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Conclusion

• Granulocyte transfusion therapy was useful and
  safe for severe neutropenic patients with
  infection, especially given at early stage.
• Controlled clinical trials will be necessary to
  establish its indications and efficacy in the
  management of infectious complications in
  neutropenic patients.

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Protocol Proposal
Neutropenic fever gt 10 days w/ inclusion criteria
randomization
Baseline APACHE/SAPS
Permitting cross-over
Treatment arm
Control arm
daily APACHE/SAPS
30 day mortality Overall mortality
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