IV pulse cyclophosphamide for vasculitis- Addenbrooke's Hospital

CYC is given as intravenous pulses at weeks 0, 2, 4 and then every 3 weeks until the remission is reached at 3 - 6 months from start of therapy (max 10 doses min 6).

a) CYC may be stopped from 3 months onward provided patient in remission (BVAS 0 for 2 consecutive study assessments). After completion of CYC, AZA to be commenced.

b) Reductions for renal function and age according to table above.

c) Maximum CYC pulse is 1.2g.

d) Upon completion of CYC course AZA to be commenced.

e) Dissolve CYC in water for injection, then dilute in saline 0.9% 500 ml and administer as IV infusion over one hour.

f) Mesna is optional and will be administered orally in the same dose in mg as CYC in mg either from IV vials or in the form of tablets on days when CYC is administered. (If it has to be administered IV reduce mesna dose to 60% of the CYC dose).

g) Prevention of emesis: the choice of antiemetic drugs to cover the CYC pulses should follow local practice. Ondansetron is suitable for this indication.

h)Check FBC on day of pulse or previous day.
If WBC prior to pulse < 4 x 109/L, then postpone pulse until WBC > 4 x 109/L, while checking WBC at least weekly. Reduce dose of pulse by 25%. With any further episodes of leucopenia, make equivalent dose reduction.

i) Check FBC between days 10 and 14 after a pulse. If the leucocyte nadir (i.e. the lowest leucocyte count between two CYC pulses) is < 3 x 109/L, even if the WBC just previous to the next pulse is > 4 x 109/L, then reduce the dose of the next pulse by:
j) leucocyte nadir 1 - 2 x 109/L reduce CYC dose of last pulse by 40 % of previous dose.
k) leucocyte nadir 2 - 3 x 109/L reduce CYC dose of last pulse by 20 % of previous dose.

l) If in remission by three months, or between three and six months, may switch to AZA 2mg/kg/day.

Anti-inflammatory cytokines

A general term for those immunoregulatory cytokines that counteract various aspects of inflammation, for example cell activation or the production of pro-inflammatory cytokines, and thus contribute to the control of the magnitude of the inflammatory responses in vivo.
These mediators act mainly by the inhibition of the production of pro-inflammatory cytokines or by counteracting many biological effects of pro-inflammatory mediators in different ways.
The major anti-inflammatory cytokines are:-
IL4,
IL10, and
IL13.

Other anti-inflammatory mediators include
IL16,
IFN-alpha,
TGF-beta,
IL1ra,
G-CSF,
soluble receptors for TNF or IL6.

Proinflammatory Cytokines

A general term for those immunoregulatory cytokines that favour inflammation.
The major pro-inflammatory cytokines that are responsible for early responses are:-
IL1-alpha, IL1-beta,
IL6, and
TNF-alpha.

Other pro-inflammatory mediators include:-
LIF,
IFN-gamma,
OSM,
CNTF,
TGF-beta,
GM-CSF,
IL11,
IL12,
IL17,
IL18,
IL8 and a variety of other chemokines that chemoattract inflammatory cells.

These cytokines either act as endogenous pyrogens (IL1, IL6, TNF-alpha), up-regulate the synthesis of secondary mediators and pro-inflammatory cytokines by both macrophages and mesenchymal cells (including fibroblasts, epithelial and endothelial cells), stimulate the production of acute phase proteins, or attract inflammatory cells.

The net effect of an inflammatory response is determined by the balance between pro-inflammatory and anti-inflammatory cytokines. It should be noted that the common and clear-cut classification of cytokines as either pro anti-inflammatory or pro-inflammatory may be misleading. The type, duration, and also the extent of cellular activities induced by one particular cytokine can be influenced considerably by the nature of the target cells, the micro-environment of a cell, depending, for example, on the growth and activation state of the cells, the type of neighboring cells, cytokine concentrations, the presence of other cytokines, and even on the temporal sequence of several cytokines acting on the same cell.

Elispot

The Enzyme-linked immunosorbent spot (ELISPOT) assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983.[1]

The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. ELISPOT assays were originally developed to enumerate B cells secreting antigen-specific antibodies, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.

By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible before are now relatively easy. This exceptional sensitivity is in part because the product is rapidly captured around the secreting cell: before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much more sensitive than conventional ELISA measurements. Limits of detection are below 1/100,000 rendering the assay uniquely useful for monitoring antigen-specific responses, applicable to a wide range of areas of immunology research, including cancer, transplantation, infectious disease, and vaccine development. The assay has gained a recent increase in popularity, especially as a surrogate measure for CTL responses, in large part because it is both reliable and highly sensitive.

While ELISPOT assay techniques have existed for more than two decades now advancements are still being made in the assay. Modern ELISPOT analysis is typically performed using ELISPOT readers, which employ computer vision techniques to enumerate the actively producing cells. This allows much of the analysis process to be automated, and permits a greater level of accuracy than what can be achieved using manual inspection.

Etanercept- from uptodate

Etanercept: Drug information


SPECIAL ALERTS
Tumor Necrosis Factor (TNF) Blockers and Malignancy Risk - June 5, 2008

The U.S. Food and Drug Administration (FDA) issued an Early Communication to healthcare professionals regarding a possible association between TNF blocker (adalimumab, certolizumb pegol, etanercept, and infliximab) use and the development of malignancies in children and young adults. Over the last 10 years, the FDA has received ~30 reports of cancer in children or young adults who had been treated with TNF blockers prior to the age of 18 years. TNF blockers were given for the treatment of Juvenile Idiopathic Arthritis (JIA [formerly termed Juvenile Rheumatoid Arthritis]), Crohn's disease, or other indications in combination with other immunosuppressive medications (eg, azathioprine, 6-mercaptopurine or methotrexate). Approximately half of the reported cancers were lymphomas (Hodgkin's and non-Hodgkin's), which are cancers involving the cells of the immune system.

TNF blockers work by suppressing the immune system. The prescribing information for each TNF blocker contains warnings regarding the possible association of malignancy development with use. Malignancies may not be detected in short-term studies; long-term studies are necessary to identify the impact of TNF blocker therapy on malignancy development. The manufacturers of the four TNF blockers available in the U.S. are being asked by the FDA to provide information regarding all cases of cancer reported in children taking TNF blockers. The FDA is expected to report its findings in approximately 6 months, after completing a safety review and evaluation.

Additional information is available at http://www.fda.gov/medwatch/safety/2008/safety08.htm#TNF

Etanercept (Enbrel®): Revised Prescribing Information With the Addition of a Boxed Warning Concerning the Risk of Infection, Including Tuberculosis - May 2008

These product labeling changes have previously been incorporated into the etanercept Lexi-Comp monograph.

The FDA MedWatch alert can be found at: http://www.fda.gov/medwatch/safety/2008/safety08.htm#Enbrel

U.S. BRAND NAMES — Enbrel®

PHARMACOLOGIC CATEGORY
Antirheumatic, Disease Modifying
Tumor Necrosis Factor (TNF) Blocking Agent

DOSING: ADULTS
Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis: SubQ:
Once-weekly dosing: 50 mg once weekly
Twice-weekly dosing: 25 mg given twice weekly (individual doses should be separated by 72-96 hours)

Plaque psoriasis:
Initial: 50 mg twice weekly, 3-4 days apart (starting doses of 25 or 50 mg once weekly have also been used successfully); maintain initial dose for 3 months
Maintenance dose: 50 mg once weekly

DOSING: PEDIATRIC — Juvenile idiopathic arthritis: Children 2-17 years: SubQ:

(For additional information see "Etanercept: Pediatric drug information")

Once-weekly dosing: 0.8 mg/kg (maximum: 50 mg/dose) once weekly

Twice-weekly dosing: 0.4 mg/kg (maximum: 25 mg/dose) twice weekly (individual doses should be separated by 72-96 hours)

DOSING: ELDERLY — SubQ: Refer to adult dosing. Although greater sensitivity of some elderly patients cannot be ruled out, no overall differences in safety or effectiveness were observed.

DOSAGE FORMS — Excipient information presented when available (limited, particularly for generics); consult specific product labeling.

Injection, powder for reconstitution:
Enbrel®: 25 mg [contains sucrose 10 mg; diluent contains benzyl alcohol]

Injection, solution [preservative free]:
Enbrel®: 50 mg/mL (0.51 mL, 0.98 mL) [contains sucrose 1%; natural rubber/natural latex in packaging]

DOSAGE FORMS: CONCISE
Injection, powder for reconstitution:
Enbrel®: 25 mg

Injection, solution [preservative free]:
Enbrel®: 50 mg/mL (0.51 mL, 0.98 mL)

GENERIC EQUIVALENT AVAILABLE — No

ADMINISTRATION — Administer subcutaneously. Rotate injection sites. New injections should be given at least one inch from an old site and never into areas where the skin is tender, bruised, red, or hard. Note: If the physician determines that it is appropriate, patients may self-inject after proper training in injection technique.

Powder for reconstitution: Follow package instructions carefully for reconstitution. The maximum amount injected at any single site should not exceed 25 mg.

Solution for injection: May be allowed to reach room temperature prior to injection.

USE — Treatment of moderately- to severely-active rheumatoid arthritis (RA); moderately- to severely-active polyarticular juvenile idiopathic arthritis (JIA); psoriatic arthritis; active ankylosing spondylitis (AS); moderate-to-severe chronic plaque psoriasis

ADVERSE REACTIONS SIGNIFICANT — Percentages reported for adults except where specified.

>10%:
Central nervous system: Headache (17%; children 19%)
Gastrointestinal: Abdominal pain (5%; children 19%), vomiting (3%; children 13%)
Local: Injection site reaction (14% to 37%; erythema, itching, pain or swelling)
Respiratory: Respiratory tract infection (upper; 12% to 29%), rhinitis (12% to 16%)
Miscellaneous: Infection (35%; children 63%), positive ANA (11%), positive antidouble-stranded DNA antibodies (15% by RIA, 3% by Crithidia luciliae assay)

≥3% to 10%:
Cardiovascular: Edema (2% to 8%)
Central nervous system: Dizziness (7%)
Dermatologic: Rash (5%)
Gastrointestinal: Dyspepsia (4%), nausea (children 9%)
Neuromuscular & skeletal: Weakness (5%)
Respiratory: Pharyngitis (7%), respiratory disorder (5%), sinusitis (3%), cough (6%)

<3%, postmarketing, and/or case reports: Abscess, adenopathy, allergic reactions, alopecia, anemia, angioedema, anorexia, aplastic anemia, appendicitis, aseptic meningitis, bursitis, cerebral ischemia, chest pain, cholecystitis, coagulopathy; demyelinating CNS disorders (suggestive of multiple sclerosis, transverse myelitis, or optic neuritis); deep vein thrombosis, depression, diarrhea, dyspnea, erythema multiforme, fatigue, fever, flushing, flu-like syndrome, gastrointestinal hemorrhage, heart failure, hepatitis (autoimmune), hydrocephalus (with normal pressure), hyper-/hypotension; infections (bacterial, fungal, protozoal, viral); interstitial lung disease, intestinal perforation, joint pain, leukopenia, lupus-like syndrome, lymphadenopathy, malignancies (including lymphoma), membranous glomerulopathy, MI, mouth ulcer, multiple sclerosis, myocardial ischemia, neutropenia, ocular inflammation, optic neuritis, pancytopenia, pancreatitis, paresthesia, polymyositis, pruritus, psoriasis exacerbation, pulmonary disease, pulmonary embolism, renal calculus, sarcoidosis, seizure, stroke, Stevens-Johnson syndrome, subcutaneous nodules, taste disturbances, thrombocytopenia, thrombophlebitis, toxic epidermal necrolysis, transaminases increased, tuberculosis, tuberculous arthritis, urinary tract infection, urticaria, vasculitis (cutaneous), weight gain, xerophthalmia, xerostomia

CONTRAINDICATIONS — Hypersensitivity to etanercept or any component of the formulation; patients with sepsis (mortality may be increased); active infections (including chronic or local infection)

WARNINGS / PRECAUTIONS
Boxed warnings:

* Infections: See "Concerns related to adverse effects" below.

* Tuberculosis: See "Concerns related to adverse effects" below.

Concerns related to adverse effects:

* Anaphylaxis/hypersensitivity reactions: Allergic reactions may occur, but anaphylaxis has not been observed. If an anaphylactic reaction or other serious allergic reaction occurs, administration should be discontinued immediately and appropriate therapy initiated.
* Autoimmune disorder: Positive antinuclear antibody titers have been detected in patients (with negative baselines). Rare cases of autoimmune disorder, including lupus-like syndrome or autoimmune hepatitis, have been reported; monitor and discontinue if symptoms develop.
* Hepatitis B: Rare reactivation of hepatitis B has occurred in chronic carriers of the virus; evaluate prior to initiation and during treatment in patients at risk for hepatitis B infection.
* Infections: [U.S. Boxed Warning]: Serious and potentially fatal infections have been reported including bacterial sepsis and tuberculosis. Discontinue administration if patient develops a serious infection or sepsis. Caution should be exercised when considering the use in patients with chronic infection, history of recurrent infection, or predisposition to infection (such as poorly-controlled diabetes). Do not give to patients with an active chronic or localized infection. Patients should be educated about the symptoms of infection and closely monitored for signs and symptoms while undergoing treatment.
* Malignancy: Use may affect defenses against malignancies; impact on the development and course of malignancies is not fully defined. As compared to the general population, an increased risk of lymphoma has been noted in clinical trials; however, rheumatoid arthritis has been previously associated with an increased rate of lymphoma.
* Tuberculosis: [U.S. Boxed Warning]: Tuberculosis (disseminated or extrapulmonary) has been reported in patients receiving etanercept; both reactivation of latent infection and new infections have been reported. Patients should be evaluated for latent tuberculosis infection with a tuberculin skin test prior to starting therapy. Treatment of latent tuberculosis should be initiated before therapy is used. Some patients who tested negative prior to therapy have developed active infection; monitor for signs and symptoms of tuberculosis in all patients.

Disease-related concerns:

* Demyelinating CNS disease: Use with caution in patients with pre-existing or recent onset CNS demyelinating disorders; rare cases of new onset or exacerbation of CNS demyelinating disorders have occurred; may present with mental status changes and some may be associated with permanent disability. Optic neuritis, transverse myelitis, multiple sclerosis, and new onset or exacerbation of seizures have been reported.
* Heart failure: Use with caution in patients with heart failure or decreased left ventricular function; worsening and new-onset heart failure has been reported.
* Hematologic disorders: Use with caution in patients with a history of significant hematologic abnormalities; has been associated with pancytopenia and aplastic anemia (rare cases in postmarketing experience). Patients must be advised to seek medical attention if they develop signs and symptoms suggestive of blood dyscrasias; discontinue if significant hematologic abnormalities are confirmed.
* Wegener's granulomatosis: Use is not recommended for use in patients with Wegener's granulomatosis who are receiving immunosuppressive therapy due to higher incidence of noncutaneous solid malignancies.

Concurrent drug therapy issues:

* Anakinra: Due to higher incidence of serious infections, should not be used in combination with anakinra unless no satisfactory alternatives exist, and then only with extreme caution.

Special populations:

* Pediatrics: Safety and efficacy have not been established in children <2 years of age.
* Varicella virus exposure: Patients with a significant exposure to varicella virus should temporarily discontinue therapy; treatment with varicella zoster immune globulin should be considered.

Dosage form specific issues:

* Latex: Some dosage forms may contain dry natural rubber (latex).

Other warnings/precautions:

* Immunizations: Patients should be brought up to date with all immunizations before initiating therapy. Live vaccines should not be given concurrently; there is no data available concerning secondary transmission of live vaccines in patients receiving therapy.

RESTRICTIONS
An FDA-approved patient medication guide is available and must be distributed when dispensing an outpatient prescription (new or refill) where this medication is to be used without direct supervision of a healthcare provider. Medication guides are available at http://www.fda.gov/cder/Offices/ODS/medication_guides.htm.

DRUG INTERACTIONS
Abatacept: Anti-TNF Agents may enhance the adverse/toxic effect of Abatacept. An increased risk of serious infection during concomitant use has been reported. Risk D: Consider therapy modification

(For additional information: Launch Lexi-Interact™ Drug Interactions Program )

Anakinra: Anti-TNF Agents may enhance the adverse/toxic effect of Anakinra. An increased risk of serious infection during concomitant use has been reported. Risk X: Avoid combination

Cyclophosphamide: Etanercept may enhance the adverse/toxic effect of Cyclophosphamide. An increased risk of solid cancer development may be present. Risk D: Consider therapy modification

Echinacea: May diminish the therapeutic effect of Immunosuppressants. Risk D: Consider therapy modification

Natalizumab: Immunosuppressants may enhance the adverse/toxic effect of Natalizumab. Specifically, the risk of concurrent infection may be increased. Risk X: Avoid combination

Rilonacept: Anti-TNF Agents may enhance the adverse/toxic effect of Rilonacept. Risk X: Avoid combination

Trastuzumab: May enhance the neutropenic effect of Immunosuppressants. Risk C: Monitor therapy

Vaccines (Inactivated): Immunosuppressants may diminish the therapeutic effect of Vaccines (Inactivated). Risk C: Monitor therapy

Vaccines (Live): Immunosuppressants may enhance the adverse/toxic effect of Vaccines (Live). Vaccinal infections may develop. Immunosuppressants may also decrease therapeutic response to vaccines. Risk X: Avoid combination

ETHANOL / NUTRITION / HERB INTERACTIONS — Herb/Nutraceutical: Echinacea may decrease the therapeutic effects of etanercept (avoid concurrent use).

PREGNANCY RISK FACTOR — B (show table)

PREGNANCY IMPLICATIONS — Developmental toxicity studies performed in animals have revealed no evidence of harm to the fetus. There are no studies in pregnant women; this drug should be used during pregnancy only if clearly needed. A pregnancy registry has been established to monitor outcomes of women exposed to etanercept during pregnancy (877-311-8972).

LACTATION — Excretion in breast milk unknown/not recommended

BREAST-FEEDING CONSIDERATIONS — It is not known whether etanercept is excreted in human milk. Because many immunoglobulins are excreted in human milk and the potential for serious adverse reactions exists, a decision should be made whether to discontinue nursing or to discontinue the drug, taking into account the importance of the drug to the mother.

PRICING — (data from drugstore.com)
Kit (Enbrel)
25 mg (4): $748.92

Solution (Enbrel)
50 mg/mL (3.92): $1471.69

Solution (Enbrel SureClick)
50 mg/mL (3.92): $1541.98

MONITORING PARAMETERS
Signs and symptoms of infection (prior to and during therapy); latent TB screening prior to therapy initiation

CANADIAN BRAND NAMES — Enbrel®

INTERNATIONAL BRAND NAMES — Enbrel (AR, AT, AU, BE, BG, BR, CH, CN, CO, CR, CZ, DE, DK, ES, FI, FR, GB, GR, GT, HK, HN, IE, IL, IN, IT, KP, MX, MY, NI, NL, NO, PA, PE, PH, PL, PT, RU, SE, SG, SV, TH, TR, VE)

MECHANISM OF ACTION — Etanercept is a recombinant DNA-derived protein composed of tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. Etanercept binds tumor necrosis factor (TNF) and blocks its interaction with cell surface receptors. TNF plays an important role in the inflammatory processes and the resulting joint pathology of rheumatoid arthritis (RA), polyarticular-course juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), and plaque psoriasis.

PHARMACODYNAMICS / KINETICS
Onset of action: ~2-3 weeks; RA: 1-2 weeks

Half-life elimination: RA: SubQ: 72-132 hours

Time to peak: RA: SubQ: 35-103 hours

Excretion: Clearance: Children: 45.9 mL/hour/m2; Adults: 89 mL/hour (52 mL/hour/m2)

PATIENT INFORMATION — If self-injecting, follow instructions for injection and disposal of needles exactly. If redness, swelling, or irritation appears at the injection site, contact prescriber. Do not have any vaccinations while using this medication without consulting prescriber first. You may experience headache or dizziness (use caution when driving or engaging in tasks requiring alertness until response to drug is known). If stomach pain or cramping, unusual bleeding or bruising, persistent fever, paleness, blood in vomitus, stool, or urine occurs, stop taking medication and contact prescriber immediately. Also immediately report skin rash, unusual muscle or bone weakness, or signs of respiratory flu or other infection (eg, chills, fever, sore throat, easy bruising or bleeding, mouth sores, unhealed sores).

Infliximab administration protocol

Protocol for Infliximab Infusion (Adults)


Indications and Dosage:
Rheumatoid arthritis, used in combination with methotrexate, where response to at least two DMARDS has been inadequate (NICE guidance).
• 3mg/kg IV infusion, followed by additional 3mg/kg at weeks 0, 2 and 6 weeks, then every 8 weeks depending on response/need. Clinical response is usually achieved within 12 weeks. Continued therapy should be reconsidered in patients who show no evidence of therapeutic benefit within this time.

Severe, active Crohn’s disease – treatment of severe, active disease in patients who have not responded despite a full and adequate course of therapy with a corticosteroid or immunosuppressant; or who are intolerant or have medical contraindications for such therapies.
• 5mg/kg IV infusion at 0, 2 and 6 weeks. In patients who respond alternative strategies for continued treatment are: • Maintenance: repeat infusions of 5mg/kg every 8 weeks depending on response/need or • Readministration: 5mg/kg infusion if signs and symptoms recur within 16 weeks of last infusion

Fistulising, active Crohn’s disease - treatment of fistulising, active Crohn’s disease, in patients who have not responded, despite full and adequate course of conventional treatment.
• initial 5mg/kg IV infusion, followed with additional 5mg/kg infusion doses at 2 and 6 weeks after the first infusion. If a patient does not respond after 3 doses, no additional treatment with infliximab should be given. In patients who respond alternative strategies for continued treatment are: • Additional infusions of 5mg/kg every 8 weeks or • Readministration if signs and symptoms of the disease recur within 16 weeks of last infusion followed by infusions of 5mg/kg every 8 weeks.

Ulcerative colitis - treatment of moderately to severely active ulcerative colitis in patients who have had an inadequate response to conventional therapy including corticosteroids and 6-mercaptopurine or azathioprine, or who are intolerant to or have medical contraindications for such therapies.
• 5 mg/kg given as an intravenous infusion over a 2 hour period followed by additional 5 mg/kg infusion doses at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. • Clinical response is usually achieved within 14 weeks of treatment, i.e. three doses. • Continued therapy should be carefully reconsidered in patients who show no evidence of therapeutic benefit within this time period.

Ankylosing spondylitis – in patients who have severe axial symptoms, elevated serological markers of inflammatory activity and who have responded inadequately to conventional therapy.
• 5mg/kg IV infusion followed by additional 5mg/kg infusion at 2 and 6 weeks after the first infusion, then every 6-8 weeks depending on response/need thereafter. If a patient does not respond by 6 weeks (ie after 2 doses), no additional treatment with infliximab should be given.

Psoriatic arthritis, in combination with methotrexate, for treatment of active and progressive psoriatic arthritis in patients who have responded inadequately to DMARDs. • 5mg/kg IV infusion followed by 5mg/kg infusion at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter depending on response/need.


Pretreatment:

Patients should be supplied with the package leaflet and special Alert card.

Contraindications:

• patients with TB or other severe infections such as sepsis, abcesses and opportunistic infections
• patients with moderate of severe heart failure
• hypersensitivity to infliximab, other murine proteins or any of the excipients




Special Warnings and Precautions for Use:
Infusion reactions • Associated with acute infusion-related reactions (eg wheezing, hypotension, pallor, nausea, anaphylactic shock and delayed hypersensitivity) which may develop within seconds or within a few hours following infusion. If this occurs then stop infusion immediately and refer to doctor.
• For severe reactions patients may require treatment with IV hydrocortisone, nebulised salbutamol and chlorphenamine.
• For mild reactions (including: drop in systolic BP < 40mmHg normal blood pressure) the infusion should be temporarily stopped until symptoms subside. Following discussion with a doctor the infusion may be restarted at a slower rate. Some patients may be rechallenged after several weeks.
• Hydrocortisone and/or paracetamol 30 mins before infliximab can prevent mild and transient effects. Infections
• Monitor closely for infections, including TB, before, during and up to 6 months after treatment



When to Withold Treatment

• Presentation of signs and symptoms of intercurrent infection. This includes upper respiratory tract infection and skin ulcer.
• Worsening of coexisting illness such as CCF or diabetes
• Suspected malignancy
• Persistent hypotension (systolic < 100mmHg)
• Impending Surgery (4 week gap for standard procedures; 8 week gap for high sepsis- risk surgery) – restart after wound healing



Exclude Infection Prior to Each Administration:

• Take a complete history and examination
• Monitor: • temperature, • urinalysis – refer to doctor if positive for blood or protein
• Send for the following bloodtests: FBC, ESR, U&Es, LFTs, CRP
• Additional tests for RA patients: Rheumatoid factor, ANA-dsDNA


Administration and Monitoring:

• Infliximab is obtained from the Aseptic Dispensing Unit (ADU), Pharmacy.
24 hour notice is required.
• IV Hydrocortisone 100-200mg stat prior to infliximab - not always necessary, there is evidence to suggest that this reduces the incidence of infliximab antibodies. Check with the prescriber.
• Insert venflon and flush with normal saline.
Monitor blood pressure, pulse and temperature every 30 minutes during the infusion and observation periods.
Patients should be observed for at least 1-2 hours post infusion for acute infusion related reactions.
• Infliximab is given in 250ml of sodium chloride 0.9%, and should be infused over a 2 hour period.
Occasionally there are patient specific deviations from this regimen.
The drug can be given through a normal giving set, via a 1.2-micron low-protein binding filter.
Filters can be ordered from supplies: "1.2micron filter lipid solutions priming volume 0.2cc male/female connectors" Medex code MX1483 Supplier = NHS Logistics, code FTC192 Rheumatoid arthritis patients – for carefully selected patients who have received 3 initial infusions over a 2 hour period, subsequent infusions can be infused over a period of not less than 1 hour, and patients observed for 1 hour afterwards.


Side Effects and Toxicity:

Recognised side effects of treatment include flu-like symptoms, headache, transient fever, gastrointestinal upset and skin rashes. Please consult the infliximab data sheet available on-line at www.medicines.org.uk for a complete list. Rarely patients have become ANA positive and developed lupus-like syndromes while receiving treatment. Adverse reactions should be reported to the CSM using the “Yellow Cards” in the usual manner.

Plasmapheresis Protocol- Hospital Kuala Lumpur

1-2 plasma volume/session
PV= (1-Hct)(b+cW)
Hct= Hematocrit
b=1530 for males, 864 for females
c=41 for males, 47.2 for females
W= lean body weight

Rough estimation: at Hct 45.
Plasma Volume=40ml/kg (BW)
Usually 5-7 sessions of plasmapheresis is required exchanges is usu done 24 hours apart
Replacement flluid- 5% human albumin
Monitor VS every 15 min
Measure PT,PTT and pre&post procedure RP, LFT, FBC

Anticoagulation: Heparin 50u/kg stat than 1000u/h
target ACT 1.5-2.0X normal
adjust dose accordingly
stop heparin 30min prior to end of procedure

To avoid hemolysis, PE must be done at
TMP 50-60mmHg
Qb 100-150mls/min

Complications of PE
Hypotension- bld loss, decrease oncotic pressure
Bleeding d/t reduction in coagulation factors
Edema- reduction of intravascular oncotic pressure
Loss of cellular elements
Ethylene Oxide hypersensitivity

Related to anticoagulation
Bleeding- heparin related
Hypocalcemia leading to arrhythmias/ hypotension/ numbness/ tingling sensation

CD86

Also called B7-2

T cells need two signals for activation - the first signal is antigen peptide presented on MHC class II through the T cell receptor

The second (costimulatory) signal is delivered by CD80 or CD86, expressed on surface of antigen presenting cells, which interact with either CD28 or CD152 (CTLA-4)

CD80 and CD86 appear to have opposing functions on regulatory T cells (J Immunol 2004;172:2778)

Polymorphisms are associated with liver transplant acceptance (Transpl Immunol 2005;15:69) and systemic sclerosis (Int J Immunogenet 2006;33:155)

Increased expression may cause excessive antigen presentation in fulminant hepatic failure as an early step in its pathogenesis before the onset of tissue damage (Am J Pathol 1999;154:1711)

High circulating soluble levels are poor prognostic factor in myeloma (Br J Haematol 2006;133:165); are associated with severe asthma (Thorax 2004;59:870)

Receptor for some adenovirus species (Virus Res 2006;122:144)

Associated with H. pylori dependent early stage high grade MALT lymphoma of stomach (World J Gastroenterol 2005;11:4357)

Uses: no significant clinical use by pathologists

Diagram: costimulatory signal

Micro images: inflamed skin; fulminant hepatic failure; chronic HBV infection of liver; liver sinusoidal endothelial cells; thyroid carcinoma (E-H); normal esophagus; esophageal carcinoma

early stage high grade gastric MALT lymphoma - H. pylori dependent case (CD86+); H. pylori independent case (CD86-)

Positive staining (normal): interdigitating dendritic cells in T zones of secondary lymphoid organs, Langerhans cells, peripheral blood dendritic cells, memory B cells, germinal center B cells, monocytes, endothelial cells, activated T cells

Positive staining (disease): AML (29%, Clin Cancer Res 2005;11:5708), ulcerative colitis (100%, Dig Dis Sci 2004;49:1738)

Negative staining: immature dendritic cells

References: OMIM 601020

CD43

Also called leukosialin, sialophorin

Appears to be important for immune function and may be part of a physiologic ligand-receptor complex involved in T-cell activation

Serves as a ligand for E-selectin on T cells and may regulate T cell trafficking (Blood 2006;107:1421, J Immunol 2005;175:8042)

Defective expression in T cells of males with Wiskott-Aldrich syndrome (OMIM 301000)

Uses: pan T cell marker, diagnosis of granulocytic sarcoma (J Clin Pathol 2005;58:211), classify T cell and low grade B cell lymphoma subtype, differentiate pulmonary MALT lymphoma (CD20+ CD43+) from lymphoid hyperplasia (CD43 neg, AJSP 2002;26:76)

Micro images: anaplastic large B cell lymphoma #1 of breast (figure c); #2-post transplant tumor of stomach (figure D); #3-AIDS associated; blastic NK lymphoma; #1 of breast (figure 6); atypical SLL/CLL

Positive staining (normal): most T cells, activated B cells, B cells in terminal ileum (Appl Immunohistochem Mol Morphol 2005;13:138), plasma cells (Scand J Immunol. 1991 Feb;33(2):211-8.), NK cells, granulocytes, monocytes, megakaryocytes, erythroid cells, hematogones (Br J Haematol 2005;128:820), Langerhans cells, brain, thymocytes, some macrophages, platelets (weak)

Positive staining (disease): T/NK cell lymphomas - anaplastic large cell (variable), blastic NK, hepatosplenic gamma-delta T cell, lymphoplasmacytic (variable), NK/T cell-nasal type (96%, Hum Path 2004;35:86), peripheral T cell, subcutaneous panniculitis-like, CD4+ CD56+ lineage negative malignancies (AJSP 2005;29:1274)

B cell lymphomas - ALL (most), Burkitt’s (almost all), Burkitt’s-like (almost all, AJSP 2005;29:1652), lymphoblastic (variable), mantle cell (100%, AJCP 2003;119:218), marginal zone (variable), nodal marginal zone (variable, AJSP 2003;27:762), plasmablastic, SLL/CLL (AJCP 1999;112:319)

other - AML, granulocytic sarcoma, hemangioma, Langerhans cell histiocytosis, mast cell disease (AJSP 2000;24:703), plasmacytoma; early colonic adenoma (Oncol Rep 2004;11:327)

Negative staining: colonic epithelium, follicular lymphoma, Hodgkin’s lymphoma, lymphoepithelioma-like thymic carcinoma, primary effusion lymphoma, splenic marginal zone lymphoma

References: OMIM 182160

CD23

Also known as low affinity IgE receptor, Fc fragment of IgE receptor, FCER

A type C lectin that can be secreted

After physiologic germinal cell development, the follicular dendritic cell meshwork expands and follicular dendritic cells in the light zone of the germinal center become CD23 positive

CD23 acts as a B cell growth and activation factor, promoting differentiation into plasma cells

Regulates IgE synthesis through CD21 and IgE binding (J Exp Med 2005;202:751), and mediates IgE related immune responses (Clin Rev Allergy Immunol 2005;29:61)

Shows variability in flow cytometry expression between specimens from same patient (AJCP 2002;117:615)

CD21, CD23 and CD35 are dendritic cell markers

High expression on B cells in peripheral blood is associated with bullous pemphigoid (J Dermatol Sci 2004;35:53)

CD23 antibodies may decrease adherence of Plasmodium falciparum-infected erythrocytes (Cell Microbiol 2004;6:839)

Mantle cell lymphoma: usually CD23 negative, but rarely/often has CD23 present with dim intensity by flow cytometry, AJCP 2003;120:760 / AJCP 2001;116:893; CD23+ mantle cell cases have high cyclin D1 levels, AJCP 2002;117:237); rarely has CD23+ cells in peripheral blood (AJCP 2002;118:758)

Uses: differentiate SLL/CLL (CD23+) vs. mantle cell lymphoma or MALT lymphoma (CD23-); B cell marker, particularly for SLL/CLL, mediastinal large B cell lymphoma and lymphoplasmacytic lymphoma; distinguish nodal mantle cell lymphoma from follicular lymphoma by identifying a disrupted follicular dendritic cell pattern (Int J Surg Pathol 2005;13:73), may identify prognostically favorable cases of diffuse large B cell lymphoma (Clin Cancer Res 2003;9:722), high soluble CD23 is associated with aggressive disease and poorer prognosis in CLL (Leuk Lymphoma 2002;43:549, Clin Lab Haematol 2006;28:30)

Micro images: angioimmunoblastic T cell lymphoma (figure F: CD23 highlights extrafollicular meshworks of follicular dendritic cells); CD23 negative MALT lymphoma with CD23+ follicular centers; CD23 negative mantle cell lymphoma with CD23+ follicular center

contributed by Leica Microsystems, Biosystems Division: normal tonsil; follicular lymphoma

Positive staining (normal): activated mature B cells expressing IgM or IgD (particularly mantle cells), activated monocytes / macrophages, T cell subsets, platelets, eosinophils, Langerhans cells, follicular dendritic cells, intestinal epithelium (encodes IgE receptor, Gastroenterology 2005;129:928)

Positive staining (disease): B-cell CLL/SLL (almost all cases; high levels, Leuk Res 2002;26:809; atypical cases may have higher levels, AJCP 2001;116:655); follicular dendritic cell tumors (including inflammatory pseudotumor type- AJSP 2001;25:721), mediastinal large B cell lymphoma (70%, Histopathology 2004;45:619), lymphoplasmacytic lymphoma (61%, usually dim intensity by flow cytometry, AJCP 2005;124:414, Clin Lymphoma 2005;5:246), hairy cell leukemia (17%, AJCP 2006;125:251), diffuse large B cell lymphoma (16%),

Negative staining: other B cell lymphomas including Burkitt’s lymphoma, Burkitt-like lymphoma (AJSP 2005;29:1652), follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (AJSP 1999;23:59, Mod Path 1998;11:967); also most T cell lymphomas, inflammatory fibroid polyps of the gastrointestinal tract (although of dendritic cell origin, AJSP 2004;28:107), follicular dendritic cell tumor, interdigitating dendritic cell tumor

References: OMIM 151445, Hum Path 1999;30:648 (early study)

CD21

Also called CR2, C3d receptor, EBV receptor

Binds to Epstein Barr virus (EBV) and HHV8 (J Virol 2005;79:4651), breakdown products of complement component C3 (C3d), CD23 (plays a role in IgE synthesis) and possibly gamma interferon

Follicular dendritic cells produce a different isoform of CD21 than B cells (J Exp Med 1997;185:165)

CD21, CD23 and CD35 are dendritic cell markers

Hodgkin’s lymphoma demonstrates disruption of follicular dendritic cell-germinal cell clusters (evaluated by CD21 and CD23)

Note: although CD21 is the receptor for EBV, it is not necessarily expressed in EBV+ tumors

Shows variability in flow cytometry expression between specimens from same patient (AJCP 2002;117:615)

Uses: diagnose follicular dendritic cell sarcomas (AJSP 1996;20:944); assess follicular dendritic cell meshwork infrastructure (AJCP 2005;124:182, AJSP 2001;25:388), distinguish cutaneous or nodal mantle cell lymphoma from follicular lymphoma (AJSP 2001;25:732, Int J Surg Pathol 2005;13:73), distinguish splenic littoral cell angioma (CD21+ lining cells) from splenic hamartomas (CD21-, AJSP 1997;21:827), confirm that atypical cells have follicular dendritic origin in fine needle aspirates of hyaline-vascular Castleman’s disease (Diagn Cytopathol 2000;22:230)

Micro images: normal germinal center

follicular dendritic cell sarcoma - (1) liver (figure 2B); (2) figure 1: liver tumor (inset: splenic tumor); 2a/b: H&E; 2c: CD21; 2d: CD35; (3) stomach (figure 4 is CD21/CD35 cocktail)

follicular lymphoma - well organized clusters of dendritic cells in follicular lymphoma #1 (testicular)); #2 (head and neck); #3 (head and neck); peripheral T cell lymphoma resembling follicular lymphoma

Positive staining (normal): mature B cells (particularly marginal and mantle cells), follicular dendritic cells, pharyngeal and cervical epithelial cells, some thymocytes, some T cells

Positive staining (tumors): follicular dendritic cell sarcoma (AJSP 2004;28:988, AJSP 2001;25:721), hairy cell leukemia, B cell lymphomas (particularly mantle and marginal zone), hyaline vascular variant of Castleman's disease (AJSP 2002;26:662), splenic littoral cell angiomas (lining cells are CD21+, AJSP 1997;21:827), some T-ALL

Negative staining: dendritic cell neurofibroma with pseudorosettes (AJSP 2001;25:587), interdigitating dendritic cell sarcoma (AJCP 2001;115:589), histiocytic sarcoma (AJSP 2004;28:1133), inflammatory fibroid polyps of GI tract (AJSP 2004;28:107), splenic hamartomas, plasma cells

References: AJSP 2001;25:721, Mod Path 2002;15:50, OMIM 120650

CD19, CD20

CD19


Coreceptor with CD21

Earliest B cell antigen in fetal tissue

Regulates B cell development, activation and differentiation (J Immunol 2003;171:5921)

May define intrinsic and antigen receptor-induced signaling thresholds critical for clonal expansion of the B cell pool and humoral immunity (Curr Dir Autoimmun 2005;8:55)

More common in plasma cells in steroid resistant ulcerative colitis than Crohn’s disease (Virchows Arch 2006;448:412); presence of CD19+ cells in intestinal mucosa may predict long remission after infliximab (anti-TNF alpha) therapy in Crohn’s disease (Hepatogastroenterology 2005;52:1128)

Uses: diagnosis of B cells and B cell disorders; may be more sensitive than CD20 to detect B cell acute leukemias (Zhongguo Shi Yan Xue Ye Xue Za Zhi 2005;13:943), to differentiate follicular lymphoma (dimmer CD19 in CD10+ B cells by flow cytometry) from reactive hyperplasia (AJCP 2005;124:576)

Flow cytometry images: follicular lymphoma with IgG light chain restriction; biphenotypic acute leukemia with CD19 and myeloperoxidase coexpression (figure B); hairy cell leukemia variant: A-CD20 (bright) and CD22+; B-CD11c+ and CD22+; C-CD103+ and CD25 negative; D-CD19+ and FMC7+; E-kappa+; F-lambda negative

Positive staining (normal): Pre B cells, B cells (considered a pan B cell antigen); first B cell antigen after HLA-DR, follicular dendritic cells

Positive staining (disease): B cell lymphomas and leukemias but often weak/negative in follicular lymphoma or diffuse large B cell lymphoma (Histopathology 2006;48:239, Cytometry B Clin Cytom 2005;63:28), occasional myeloid leukemias (AML-AJCP 1998;109:211; AML-M0-AJCP 2001;115:876; CML blast phase-AJCP 2004;121:836), occasional anaplastic large cell lymphoma by flow cytometry (AJCP 2003;119:205)

Negative staining: plasma cells, myeloma (AJCP 2004;121:482), most T cell lymphomas, often L&H cells in lymphocyte predominant Hodgkin’s lymphoma, often post-transplant B cell lymphoproliferative disorder

References: OMIM 107265



CD20


Also called L26, membrane spanning 4 domains (MS4A1)

33kd phosphoprotein with 3 hydrophobic regions that traverse the cell membrane, creating a structure similar to an ion channel that allows for the influx of calcium required for cell activation

Initially expressed on B cells after CD19/CD10 expression and before CD21/CD22 and surface immunoglobulin expression; retained on mature B cells until plasma cell development

Delivers early signal in B cell activation, allowing resting B cells to respond to later antigens

Closely related to FMC7, which recognizes a CD20 epitope (Leukemia 2003;17:1384), particularly if there is strong CD20 expression (AJCP 2003;120:754)

Rituximab is a chimeric murine-human anti-CD20 antibody used to treat B cell lymphomas; treatment may cause selection of CD20 negative (but CD79a+) tumor subclones (AJSP 2005;29:1399)

Rituximab is also used to treat autoimmune disorders (Clin Immunol 2005;117:207), TTP/HUS (Acta Cytol 2005;19:423), ABO incompatible transplantation (Transplant Proc 2005;37:1205) and transplant rejection (Clin Transplant 2005;19:137)

Anti CD20-antibody attached to radioisotopes is also used to treat B cell lymphomas (Clin Exp Med 2006;6:1)

Case reports: CD20+ T cell lymphomas (Am J Hematol 2002;71:331, AJCP 1994;102:483, Mod Path 2001;14:105, Mod Path 2000;13:1244); rarely stains nucleoli of malignant T cells (Acta Cytol 2005;49:365), but see J Clin Pathol 2004;57:442

Uses: commonly used marker for B cells

Micro images: normal lymph node #1; #2

angioimmunoblastic T cell lymphoma (figure d)

diffuse large B cell lymphoma - bone; brain-#1; #4 (figure A); #5 (intravascular); #6 (intravascular); liver; nasal cavity (figure 3A); ovary CD20 (fig 3), CD3 (fig 4); sclerosing; skin (figure 1D); small intestine; unknown site #1-intravascular; #2-sclerosing subtype

other leukemia/lymphoma - follicular lymphoma #1; #2 (childhood); hairy cell leukemia #1; #2-variant type (figure D); Hodgkin’s lymphoma #1-lymphocyte predominant (figures C&D); #2 (figure C); #3-mostly negative Reed-Sternberg cells); lymphoplasmacytic lymphoma/ Waldenström macroglobulinemia #1 (brain-figure D); MALT lymphoma #1 of bladder; #2 of liver; #3 of lung;

SLL of colon (figure C)

post-transplantation lymphoproliferative disorder - #1; #2 (polymorphic subtype-figure C); #3 of liver: H&E, CD20, EBV

other - ectopic hamartomatous thymoma (figure C); lymphocytic mastitis: CD20+/CD3- lymphocytes (B, not T cells); immunoblastic myofibroblastic tumor (figure 2d-reactive B cells)

Additional images: intravascular large cell lymphoma (figure 2a); post-transplant lymphoproliferative disorder

Virtual slides: diffuse large B cell lymphoma

Flow cytometry images: hairy cell leukemia variant - A-CD20 (bright) and CD22+; B-CD11c+ and CD22+; C-CD103+ and CD25 negative; D-CD19+ and FMC7+; E-kappa+; F-lambda negative

Positive staining (normal): most B cells (considered a pan B cell antigen), also follicular dendritic cells

Positive staining (disease): 90% of B cell lymphomas; also B-CLL, hairy cell leukemia, spindle cell thymomas (AJSP 1992;16:988), 40% of pre B ALL/LBL; 80% of nodular lymphocyte predominant Hodgkin’s lymphoma, 20% of classic Hodgkin’s lymphoma (may be an adverse prognostic factor, Br J Haematol 2004;125:701); dimly expressed in T cells (benign and neoplastic, particularly in bone marrow, AJCP 1996;106:78, AJCP 1994;102:483), some myelomas (Mod Path 2004;17:1217, Blood 2003;102:1070)

Negative staining: non-hematopoietic cells, most T cells, plasma cells, mastocytosis

Note: staining does not work well with Bouin’s fixative

Flow cytometry: brighter expression in follicular lymphomas than normal B cells (AJCP 2005;124:576)

References: OMIM 112210, J Biol Chem 2004;279:19893 (presence in lipid rafts)

CD16

CD16a

Also known as Fc gamma receptor III A, low affinity immunoglobulin gamma Fc region receptor III-A

Receptor for the Fc portion of IgG; binds various IgG molecules, including rheumatoid factor

Mediates antibody dependent cytotoxicity of foreign cells, phagocytosis and other antibody-dependent responses; also platelet satellitism (AJCP 1995;103:740)

Affinity to ligand is regulated by glycosylation (Immunology 2003;110:335)

CD14+ CD16+ monocytes have increased capacity to produce proinflammatory cytokines such as TNF-alpha, and are elevated in various inflammatory diseases, including coronary artery disease (Thromb Haemost 2004;92:419)

Polymorphisms influence: (a) the severity but not the incidence of IgA nephropathy in Japanese patients (Nephrol Dial Transplant 2005;20:2439); (b) pathogenesis of coronary artery disease (Atherosclerosis 2005;180:277), (c) clinical response to rituximab (Cancer Res 2004;64:4664)

If target cell has class I MHC, then NK cell's killer cell inhibitory receptor (KIR) inhibits cytolysis

Note: preincubation with CD16/CD32 antibodies is commonly used to prevent nonspecific binding

Uses: NK cell and macrophage marker; to subtype leukemia/lymphoma

Diagrams: NK cell mediated cytotoxicity [CD16 / FcgammaRIII is activating receptor on NK cell]

Positive staining (normal): NK cells (10-20%), granulocytes, macrophages, T cells (reactive), immature thymocytes, placental trophoblast

Positive staining (disease): NK proliferative disorders, T cell large granular lymphocyte leukemia, hepatosplenic gamma-delta T cell lymphoma

References: OMIM 146740



CD16b

Also known as Fc gamma receptor III B, low affinity immunoglobulin gamma Fc region receptor III-B

Highly homologous to CD16a

The most common receptor for the Fc domain of IgG on leukocytes

The only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor

Bears allotypes that define the human neutrophil antigen-1 (HNA-1 and NA) system involved in major post-transfusional reactions (Tissue Antigens 2004;64:119)

Low copy number is associated with glomerulonephritis in systemic lupus erythematosus (Nature 2006;439:851)

CD16+ eosinophils are upregulated in allergic conditions (J Allergy Clin Immunol 2002;109:463)

Affinity to ligand is regulated by glycosylation (Immunology 2003;110:335)

Uses: no significant clinical use by pathologists

Positive staining: neutrophils

CD8

Also called T cell suppressor/cytotoxic cells, OKT8

Cell surface glycoprotein, member of immunoglobulin superfamily; at 2p12

Heterodimer of an alpha and a beta chain linked by two disulfide bonds; heterodimer on thymocytes and homodimer on peripheral blood T cells

MHC class I restricted receptor; binds to nonpolymorphic region of class I molecules; may increase avidity of interactions between cytotoxic T cell and target cell during antigen-specific activation

Can kill target cells by recognizing peptide-MHC complexes on them or by secreting cytokines capable of signaling through death receptors on target cell surface

CD8 alpha cells promote survival and differentiation of activated T cells into memory CD8+ T cells, which may become clonal (but not malignant) in the elderly (Immunol Rev 2005;205:170)

Contribute to initiation, progression and regulation of autoimmune responses (Curr Opin Immunol 2005;17:624)

Associated with lymphoepithelioma-like carcinoma of lung (AJSP 2002;26:715); low CD8:CD3 ratio in epidermotrophic component of lymphoid infiltrate is suggestive of mycosis fungoides (Mod Path 2003;16:857)

Uses: cytotoxic T cell marker

Drawings: (1) CD8+ T cell interaction with antigen presenting cell #1; #2; (3) destruction of viral infected cell

Micro images: (4) infiltrating lymphocytes in lymphoepithelioma-like carcinoma of cervix-figure 3; (5) T cell lymphoma (type not specified); (6) sinus lining cells in splenic hamartomas #1 are CD8+; #2 (figure 3A); (8) nodal cytotoxic T cell lymphoma; (9) mycosis fungoides; (10) intraepithelial lymphocytes in duodenum (figure 1c); (11) increased intraepithelial lymphocytes at villous tip (figure 2e)

Positive staining (normal): cortical thymocytes (70-80%), T cells (25-35% of mature peripheral T cells, mostly cytotoxic T cells); NK cells (30%, which are also CD3 negative)

Positive staining (disease): epidermotrophic lymphocytes in mycosis fungoides (AJSP 2002;26:450), subcutaneous panniculitis-like T cell lymphoma, indolent T cell lymphoblastic proliferation, sinus lining cells in splenic hemartoma, heterotopic ovarian splenoma, NK/T cell lymphoma (variable), some post-thymic T cell lymphomas, rarely mantle cell lymphoma (AJCP 1998;109:689), rarely CLL (AJCP 1994;102:212, Archives 2000;124:1361), T cell infiltrate in 30% of cases of nodular regenerative hyperplasia of liver (Hum Path 2004;35:1241)

Negative stains: adult T cell leukemia/lymphoma, littoral cell hemangioma of spleen

References: Mod Path 2002;15:1131; OMIM 186910 (alpha chain), OMIM 186730 (beta chain)

CD4

Also called OKT4

Nonpolymorphous glycoproteins belonging to immunoglobulin superfamily

Expressed on surface of T helper cells; serves as coreceptor in MHC class II-restricted antigen induced T cell activation

CD4+ CD25+ T cells maintain peripheral tolerance and prevent autoimmunity (Curr Top Microbiol Immunol 2005;293:115)

Serves as HIV receptor on T cells, macrophages, brain

Downregulated by HIV Nef protein during AIDS progression (J Virol 2003;77:11536, J Biol Chem 2003;278:33912)

Normally CD4 > CD8; in HIV patients, CD4/CD8 ratio is inverted (i.e. CD4 < CD8) and patients are at risk for opportunistic infections

Homologous to CD223

Uses: classify lymphomas and inflammatory conditions; serum levels are marker of HIV disease progression and response to therapy (CD4+ cells are killed by HIV); serum levels also increased by transient stress (AJCP 2002;117:819)

Drawings: (1) CD4+ T cell and antigen presenting cell; (2) HIV entry into T cells

Positive staining (normal): thymocytes (80-90%), T helper cells, macrophages, Langerhans cells, dendritic cells, granulocytes

Positive staining (disease): many post-thymic T cell leukemia/lymphomas, indolent T cell lymphoblastic proliferation, pityriasis lichenoides, CD4+ CD56+ hematodermic malignancies (blastic NK lymphoma), histiocytic lymphoma / sarcoma, acute myeloid leukemia (AJCP 1995;104:204), some pyothorax associated lymphomas, cutaneous lymphomatoid granulomatosis (AJSP 2001;25:1111), lymphomatoid papulosis (variable), florid antiviral inflammatory response (Mod Path 2003;16:166)

Negative staining: NK cells, T cell lymphoma with cytotoxic phenotype, hepatosplenic alpha/beta and gamma/delta lymphoma, enteropathy associated T cell lymphoma, B cell lymphoma (usually), Hodgkin’s lymphoma (usually), nonhematopoietic neoplasms

References: Cell 1985;42:93 (early article), OMIM 186940

Gating in FACS

Gating will allow you to view cells of interest by any combination of criteria that you choose. Gating does not change the intensity value assigned to an event as is the case for changes in voltage or compensation.

It simply lets you decide which data to view and which data to ignore or discard.

It is important to check that small changes in your gates don't have significant effects on your results or else your data will be prone to artifact.

When you create or format a data plot (i.e. on the Data menu select "Format Histogram" or "Format Dot Plot") you can select any of the gates that you have created.

This will filter the data and plot will only display those events which meet the gate criteria.

Gating will not discard data unless you have requested this under "Acquisiton and Storage". Gating can subsequently be changed when you analyze your data without any loss of information.

To set up a gate you first draw a "Region" using one of the tools on the tool palette (there are four geometric shapes to choose from outlined with dotted lines). Note that the "Marker bar" (designated with an M) and the quadrant maker tool next do not define regions. They are used for statistical analysis only and can't be used to filter data like the regions/gates.

Regions which are commonly employed include: PI for DNA content: FL2 area vs FL2 width. This window is useful for gaiting out apoptotic cells (lower left quadrant) and doublets (a separate cloud with increased FL2 width). FSC vs SSC: This is useful for gating out RBC, myeloid cells etc, from blood or marrow. Each region that is setup automatically defines a gate (e.g. G1 = R1). To delete regions select "Region List" under the Gates menu. To combine regions into more complex gating criteria use the "Gate List" under the Gates menu. (e.g. G5 = (R1 or R2) and R3).

What are Northern, Southern, Western, Southwestern Blots?

Southern blotting was the original of the four.
It got its name from the developer, Edwin Southern.
Western blotting was named as a sort of joke. (North, South, East, West).
The rest arose based on these first two.

Southern blotting uses gel electrophoresis for the detection of a specific DNA sequence in a sample of DNA.

Western blotting also uses gel electrophoresis but it is to detect proteins and separate them based on size and shape.

Northern blotting is for the detection of RNA sequences, and so is geared towards detecting gene expression (the technique is very similar to Southern blotting only formaldehyde is used to denature the RNA).

There is no Eastern blot, but there is a southwestern one. It is used to find where proteins (DNA binding proteins) bind to specific sequences of DNA.

Far-western blotting is a molecular biological method which is based on the technique of western blotting. While usual western blotting uses an antibody to detect a protein of interest, far-western blotting uses a non-antibody protein, which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is rather employed to detect protein:protein interactions.

By the way, they all use gel electrophoresis.





Far-Eastern blotting is different from the above. It is a technique developed in the 1990s by T. Taki and colleagues at the Cellular Technology Institute of Otsuka Pharmaceutical Co., Japan for the analysis of lipids separated by high-performance thin layer chromatography (HPTLC). The lipids are transferred from the HPTLC plate to a PVDF membrane for further analysis, for example by enzymatic or ligand binding assays[1] or mass spectrometry[2].

Cholesterol, glycerophospholipids and sphingolipids are major constituents of the cell membrane and in certain cases function as second messengers in cell proliferation, apoptosis and cell adhesion in inflammation and tumor metastasis. Far-eastern blotting was established as a method for transferring lipids from an HPTLC plate to a polyvinyledene difluoride (PVDF) membrane within a minute. Applications of this with other methods have been studied. Far-eastern blotting allows for the following techniques:

* Purification of glycosphingolipids and phospholipids.
* Structural analysis of lipids in conjunction with direct mass spectrometry.
* Binding study using various ligands such as antibodies, lectins, bacterium, viruses, and toxins, and
* Enzyme reaction on membranes.

Not only analysis of lipids but also metabolites of drugs and natural compounds from plants, and environmental hormones are possible by this method.

Alum

Alum, (IPA: /ˈæləm/) refers to a specific chemical compound and a class of chemical compounds. The specific compound is the hydrated aluminum potassium sulfate with the formula KAl(SO4)2.12H2O. The wider class of compounds known as alums have the related stoichiometry, AB(SO4)2.12H2O.


Alum is used in vaccines as an adjuvant to enhance the body's response to immunogens.

Thalidomide

WARNING

SEVERE, LIFE-THREATENING HUMAN BIRTH DEFECTS.

IF THALIDOMIDE IS TAKEN DURING PREGNANCY, IT CAN CAUSE SEVERE BIRTH DEFECTS OR DEATH TO AN UNBORN BABY. THALIDOMIDE SHOULD NEVER BE USED BY WOMEN WHO ARE PREGNANT OR WHO COULD BECOME PREGNANT WHILE TAKING THE DRUG. EVEN A SINGLE DOSE [1 CAPSULE (REGARDLESS OF STRENGTH)] TAKEN BY A PREGNANT WOMAN DURING HER PREGNANCY CAN CAUSE SEVERE BIRTH DEFECTS.

BECAUSE OF THIS TOXICITY AND IN AN EFFORT TO MAKE THE CHANCE OF FETAL EXPOSURE TO THALOMID® (thalidomide) AS NEGLIGIBLE AS POSSIBLE, THALOMID® (thalidomide) IS APPROVED FOR MARKETING ONLY UNDER A SPECIAL RESTRICTED DISTRIBUTION PROGRAM APPROVED BY THE FOOD AND DRUG ADMINISTRATION. THIS PROGRAM IS CALLED THE "SYSTEM FOR THALIDOMIDE EDUCATION AND PRESCRIBING SAFETY (S.T.E.P.S.® )".

UNDER THIS RESTRICTED DISTRIBUTION PROGRAM, ONLY PRESCRIBERS AND PHARMACISTS REGISTERED WITH THE PROGRAM ARE ALLOWED TO PRESCRIBE AND DISPENSE THE PRODUCT. IN ADDITION, PATIENTS MUST BE ADVISED OF, AGREE TO, AND COMPLY WITH THE REQUIREMENTS OF THE S.T.E.P.S.® PROGRAM IN ORDER TO RECEIVE PRODUCT.

PLEASE SEE THE FOLLOWING BOXED WARNINGS CONTAINING SPECIAL INFORMATION FOR PRESCRIBERS, FEMALE PATIENTS, AND MALE PATIENTS ABOUT THIS RESTRICTED DISTRIBUTION PROGRAM.



PRESCRIBERS

THALOMID® (thalidomide) may be prescribed only by licensed prescribers who are registered in the S.T.E.P.S.® program and understand the risk of teratogenicity if thalidomide is used during pregnancy.

Major human fetal abnormalities related to thalidomide administration during pregnancy have been documented: amelia (absence of limbs), phocomelia (short limbs), hypoplasticity of the bones, absence of bones, external ear abnormalities (including anotia, micro pinna, small or absent external auditory canals), facial palsy, eye abnormalities (anophthalmos, microphthalmos), and congenital heart defects. Alimentary tract, urinary tract, and genital malformations have also been documented.1 Mortality at or shortly after birth has been reported at about 40%.2

Effective contraception (see CONTRAINDICATIONS) must be used for at least 4 weeks before beginning thalidomide therapy, during thalidomide therapy, and for 4 weeks following discontinuation of thalidomide therapy. Reliable contraception is indicated even where there has been a history of infertility, unless due to hysterectomy or because the patient has been postmenopausal for at least 24 months. Two reliable forms of contraception must be used simultaneously unless continuous abstinence from heterosexual sexual contact is the chosen method. Women of childbearing potential should be referred to a qualified provider of contraceptive methods, if needed. Sexually mature women who have not undergone a hysterectomy or who have not been postmenopausal for at least 24 consecutive months (i.e., who have had menses at some time in the preceding 24 consecutive months) are considered to be women of childbearing potential.

Before starting treatment, women of childbearing potential should have a pregnancy test (sensitivity of at least 50 mIU/mL). The test should be performed within the 24 hours prior to beginning thalidomide therapy. A prescription for thalidomide for a woman of childbearing potential must not be issued by the prescriber until a written report of a negative pregnancy test has been obtained by the prescriber.

Male Patients: Because thalidomide is present in the semen of patients receiving the drug, males receiving thalidomide must always use a latex condom during any sexual contact with women of childbearing potential even if he has undergone a successful vasectomy.

Once treatment has started, pregnancy testing should occur weekly during the first 4 weeks of use, then pregnancy testing should be repeated at 4 weeks in women with regular menstrual cycles. If menstrual cycles are irregular, the pregnancy testing should occur every 2 weeks. Pregnancy testing and counseling should be performed if a patient misses her period or if there is any abnormality in menstrual bleeding.

If pregnancy does occur during thalidomide treatment, thalidomide must be discontinued immediately.

Any suspected fetal exposure to THALOMID® (thalidomide) must be reported immediately to the FDA via the MedWatch number at 1-800-FDA-1088 and also to Celgene Corporation at 1-888-423-5436. The patient should be referred to an obstetrician/gynecologist experienced in reproductive toxicity for further evaluation and counseling.



FEMALE PATIENTS

Thalidomide is contraindicated in WOMEN of childbearing potential unless alternative therapies are considered inappropriate AND the patient MEETS ALL OF THE FOLLOWING CONDITIONS (i.e., she is essentially unable to become pregnant while on thalidomide therapy):

* she understands and can reliably carry out instructions.
* she is capable of complying with the mandatory contraceptive measures, pregnancy testing, patient registration, and patient survey as described in the System for Thalidomide Education and Prescribing Safety (S.T.E.P.S.® ) program.
* she has received both oral and written warnings of the hazards of taking thalidomide during pregnancy and of exposing a fetus to the drug.
* she has received both oral and written warnings of the risk of possible contraception failure and of the need to use two reliable forms of contraception simultaneously (see CONTRAINDICATIONS), unless continuous abstinence from heterosexual sexual contact is the chosen method. Sexually mature women who have not undergone a hysterectomy or who have not been postmenopausal for at least 24 consecutive months (i.e., who have had menses at some time in the preceding 24 consecutive months) are considered to be women of childbearing potential.
* she acknowledges, in writing, her understanding of these warnings and of the need for using two reliable methods of contraception for 4 weeks prior to beginning thalidomide therapy, during thalidomide therapy, and for 4 weeks after discontinuation of thalidomide therapy.
* she has had a negative pregnancy test with a sensitivity of at least 50 mIU/mL, within the 24 hours prior to beginning therapy. (See PRECAUTIONS, CONTRAINDICATIONS)
* if the patient is between 12 and 18 years of age, her parent or legal guardian must have read this material and agreed to ensure compliance with the above.



MALE PATIENTS

Thalidomide is contraindicated in sexually mature MALES unless the PATIENT MEETS ALL OF THE FOLLOWING CONDITIONS:

* he understands and can reliably carry out instructions.
* he is capable of complying with the mandatory contraceptive measures that are appropriate for men, patient registration, and patient survey as described in the S.T.E.P.S.® program.
* he has received both oral and written warnings of the hazards of taking thalidomide and exposing a fetus to the drug.
* he has received both oral and written warnings of the risk of possible contraception failure and of the presence of thalidomide in semen. He has been instructed that he must always use a latex condom during any sexual contact with women of childbearing potential, even if he has undergone a successful vasectomy.
* he acknowledges, in writing, his understanding of these warnings and of the need to use a latex condom during any sexual contact with women of childbearing potential, even if he has undergone a successful vasectomy. Sexually mature women who have not undergone a hysterectomy or who have not been postmenopausal for at least 24 consecutive months (i.e., who have had menses at any time in the preceding 24 consecutive months) are considered to be women of childbearing potential.
* if the patient is between 12 and 18 years of age, his parent or legal guardian must have read this material and agreed to ensure compliance with the above.



VENOUS THROMBOEMBOLIC EVENTS

The use of THALOMID® (thalidomide) in multiple myeloma results in an increased risk of venous thromboembolic events, such as deep venous thrombosis and pulmonary embolus. This risk increases significantly when thalidomide is used in combination with standard chemotherapeutic agents including dexamethasone. In one controlled trial, the rate of venous thromboembolic events was 22.5% in patients receiving thalidomide in combination with dexamethasone compared to 4.9% in patients receiving dexamethasone alone (p = 0.002). Patients and physicians are advised to be observant for the signs and symptoms of thromboembolism. Patients should be instructed to seek medical care if they develop symptoms such as shortness of breath, chest pain, or arm or leg swelling. Preliminary data suggest that patients who are appropriate candidates may benefit from concurrent prophylactic anticoagulation or aspirin treatment.
DRUG DESCRIPTION

THALOMID® (thalidomide), α-(N-phthalimido) glutarimide, is an immunomodulatory agent. The empirical formula for thalidomide is C13H10N2O4 and the gram molecular weight is 258.2. The CAS number of thalidomide is 50-35-1.




Thalidomide is an off-white to white, odorless, crystalline powder that is soluble at 25°C in dimethyl sulfoxide and sparingly soluble in water and ethanol. The glutarimide moiety contains a single asymmetric center and, therefore, may exist in either of two optically active forms designated S-(-) or R-(+). THALOMID® (thalidomide) is an equal mixture of the S-(-) and R-(+) forms and, therefore, has a net optical rotation of zero.


THALOMID® (thalidomide) is available in 50 mg, 100 mg, 150 mg and 200 mg capsules for oral administration. Active ingredient: thalidomide. Inactive ingredients: pregelatinized starch and magnesium stearate. The 50 mg capsule shell contains gelatin, titanium dioxide, and black ink. The 100 mg capsule shell contains black iron oxide, yellow iron oxide, titanium dioxide, gelatin, and black ink. The 150 mg capsule shell contains FD&C blue #2, black iron oxide, yellow iron oxide, titanium dioxide, gelatin, and black and white ink. The 200 mg capsule shell contains FD&C blue #2, titanium dioxide, gelatin, and white ink.


Other common side effects


Thalidomide is associated with:-
drowsiness/somnolence,
peripheral neuropathy,
dizziness/orthostatic hypotension,
neutro-penia, and
HIV viral load increase.

Hypersensitivity to THALOMID® (thalidomide) and bradycardia in patients treated with thalidomide have been reported.

Somnolence, dizziness, and rash are the most commonly observed adverse events associated with the use of thalidomide.

Plasmapheresis Protocol- Addenbrooke's Hospital

Dose:-

7 sessions over 14 days
60mls/kg
5% Albumin
Use of FFP at end of procedure- at discretion of treating doctor


Other aspects of plasma-exchange applies.

Anticoagulation: Heparin 50u/kg stat than 1000u/h
target ACT 1.5-2.0X normal
adjust dose accordingly
stop heparin 30min prior to end of procedure

To avoid hemolysis, PEx must be done at
TMP 50-60mmHg
Qb 100-150mls/min

Complications of PEx
Hypotension- bld loss, decrease oncotic pressure
Bleeding d/t reduction in coagulation factors
Edema- reduction of intravascular oncotic pressure
Loss of cellular elements
Ethylene Oxide hypersensitivity

Related to anticoagulation
Bleeding- heparin related
Hypocalcemia leading to arrhythmias/ hypotension/ numbness/ tingling sensation

RITUXIMAB-Mechanism of action in NHL

Azathioprine

After hepatic conversion to 6-mercaptopurine, the cytotoxic effects of azathioprine are mediated by the impairment of purine synthesis, incorporation of purines into DNA, and impairment of the endonuclease repair activity of DNA polymerase (20). The drug is well-absorbed after oral administration and elimination requires hepatic metabolism by xanthine oxidase; an important drug interaction is with xanthine oxidase inhibitors, such as allopurinol. Lymphocyte function is reduced, B-cells more than T-cells, and there is suppression of the cellular component of the inflammatory response. The major adverse effects are nausea and vomiting, dose-dependent myelosuppression and reversible, cholestatic, hepatic toxicity. An increased incidence of malignancies, particularly lymphomas and skin cancers, has been observed with prolonged administration after organ transplantation

Cyclophosphamide

Cyclophosphamide is an inactive pro-drug, converted by the mixed function oxidase system in the liver to the alkylating agents 4-hydroxy-cyclophosphamide and phosphoramide mustard, which alkylate guanine nucleotides, thus blocking cell division (20). Bioavailability after oral administration is greater than 75%, but there are large variations between individuals in the rate of production of active metabolites. A phenotypic variation in carboxylator activity affects the production of the inactive metabolite carboxyphosphoramide from 4-hydroxy-cyclophosphamide, which may influence efficacy and toxicity. The relation of renal and hepatic failure to the production and elimination of active metabolites has not been fully determined. Bladder toxicity is caused by renal excretion of the metabolite acrolein which can cause haemorrhagic cystitis and a markedly increased risk of bladder cancer (20). Other adverse effects include nausea and vomiting, myelosuppression with neutropenia, infections due to immunosuppression (4), alopecia and infertility. Permanent ovarian failure occurs in over 50% of women after one year’s exposure and is age-related; male infertility has been less well studied. The incidence of leukaemia and /or lymphoma is increased tenfold; less common adverse effects include pulmonary fibrosis, hepatitis and the syndrome of inappropriate ADH secretion.

RITUXIMAB

Administration

* DO NOT ADMINISTER AS AN INTRAVENOUS PUSH OR BOLUS

* Premedicate before each infusion.

Administer only as an intravenous infusion
* Interrupt the infusion or slow the infusion rate for infusion reactions (see BOXED WARNING and WARNINGS AND PRECAUTIONS)
* First infusion: Initiate infusion at a rate of 50 mg/hr. In the absence of infusion toxicity, increase infusion rate by 50 mg/hr increments every 30 minutes, to a maximum of 400 mg/hr
* Subsequent infusions: Initiate infusion at a rate of 100 mg/hr. In the absence of infusion toxicity, increase rate by 100 mg/hr increments at 30-minute intervals, to a maximum of 400 mg/hr



Preparation

* RITUXAN is supplied as 100 mg/10 mL (NDC 50242-051-21) and 500 mg/50 mL (NDC 50242-053-06) solution in a single-use vial



Stability and storage

* RITUXAN vials are stable at 2°C–8°C (36°F–46°F). Do not use beyond expiration date stamped on carton. RITUXAN vials should be protected from direct sunlight. Do not freeze or shake
* RITUXAN solutions for infusion may be stored at 2°C–8°C (36°F–46°F) for 24 hours. RITUXAN solutions for infusion have been shown to be stable for an additional 24 hours at room temperature. However, since RITUXAN solutions do not contain a preservative, diluted solutions should be stored refrigerated (2°C–8°C). No incompatibilities between RITUXAN and polyvinylchloride or polyethylene bags have been observed.



WARNING: FATAL INFUSION REACTIONS, TUMOR LYSIS SYNDROME (TLS), SEVERE MUCOCUTANEOUS REACTIONS, and PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY (PML)

Infusion Reactions
Rituxan administration can result in serious, including fatal infusion reactions. Deaths within 24 hours of Rituxan infusion have occurred. Approximately 80% of fatal infusion reactions occurred in association with the first infusion. Carefully monitor patients during infusions. Discontinue Rituxan infusion and provide medical treatment for Grade 3 or 4 infusion reactions.

Tumor Lysis Syndrome (TLS)
Acute renal failure requiring dialysis with instances of fatal outcome can occur in the setting of TLS following treatment of non-Hodgkin's lymphoma (NHL) patients with Rituxan.

Severe Mucocutaneous Reactions
Severe, including fatal, mucocutaneous reactions can occur in patients receiving Rituxan.

Progressive Multifocal Leukoencephalopathy (PML)
JC virus infection resulting in PML and death can occur in patients receiving Rituxan.

Premedication:-

We give the following:-
PO Paracetamol 1g
IV Chlorpheniramine 10mg
+/- IV Methylprednisolone



We have used the following Rituximab dosing protocol for our vasculitis patients in Addenbrooke's Hospital, Cambridge.

Rituximab 1g X 2 doses 2 weeks apart
Then 1g 6 monthly for 2 years.

RITUXVAS

Background

1.1.The diseases

Wegener's granulomatosis (WG) and microscopic polyangiitis (MP) are syndromes of primary systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA) (appendix 1)(15,16,19). WG and MP share many histological features, including a necrotising glomerulonephritis which often leads to rapidly progressive renal failure. Isolated pauci-immune necrotising, cresentic glomerulonephritis, has many features to suggest that it represents a renal-limited form of vasculitis (RLV), including the presence of circulating anti-MPO or anti-Pr3 antibodies. Common histological and serological features, and a similar response to treatment, have justified a common approach to the treatment of WG, MP and RLV. These diseases are sub grouped according to severity. ‘Generalised’ vasculitis refers to systemic disease with renal involvement (creatinine up to 500) and or other imminent vital organ failure. Patients with ‘generalised’ vasculitis and those with more severe renal vasculitis (i.e. creatinine greater than 500) will be included in this trial.

1.2.Their treatment

Untreated generalised WG and MP follow a progressive course with fatal outcome due to vital organ failure (17). With the empirical introduction of corticosteroids and cytotoxic agents, five year survival has increased from under 20% to over 60% (18). Over the last decade the treatment of AASV has been standardised following the results of randomised trials. Cyclophosphamide (either daily oral or intravenous (IV) pulse) and prednisolone are used for remission induction (3 to 6 months) with longer therapy of azathioprine or methotrexate and low dose steroids to prevent disease relapse. Early systemic vasculitis may be treated with methotrexate in place of cyclophosphamide. The addition of plasma exchange improves renal recovery rates in severe renal vasculitis. Adverse events with current therapy are frequent and are the major cause of mortality. The frequency of severe adverse events (10-45%) is influenced by age and the severity of renal involvement. There is a clear need for a new safer, effective therapy for AASV (1,2,3,6). In order to address the questions of drug toxicity and frequent disease relapses, newer therapies with better safety profiles have been investigated in AASV. One such agent is rituximab, a murine/human chimeric anti CD 20 monoclonal antibody. Its use results in B cell depletion for 6-18 months (7). B cells are key factors in autoimmune diseases with roles in autoantibody production, cytokine release and antigen presentation to T-cells. The sequence of immune events that trigger AASV are not fully understood, however evidence suggests that ANCA are pathogenic (21). Thus interruption of the cell line leading to ANCA production may be beneficial in disease suppression. Rituximab is an established treatment in non-Hodgkin’s lymphoma with a good safety track record (7). Even though the individual is rendered B cell deplete, immunoglobulin levels are maintained, and infection rates are low. Recently its use in autoimmune diseases has been realised with excellent results in rheumatoid arthritis (8). Rituximab has been shown to be effective in AASV, inducing remission in patients intolerant of cyclophosphamide (5,10-12,22,26-28). Given its good safety profile and efficacy its use a first line agent in AASV needs to be investigated.


In RITUXVAS, a rituximab based regimen will be compared with a standard cyclophosphamide/azathioprine based regimen in the treatment of AASV with glomerulonephritis. In order to achieve results applicable to clinical practice, all cases from mild to severe renal failure will be included. Standard practice includes the use of oral steroids which will be applied similarly in both treatment groups. It is recognised that necrotising cresentic glomerulonephritis progresses rapidly and immediate immune suppression is required to reverse existing and spare further organ damage. The therapeutic effect of rituximab is not immediate. Therefore two initial doses of cyclophosphamide will be given along side rituximab, thus allowing early disease control. When dialysis dependant renal failure or pulmonary haemorrhage occur, plasma exchange or intravenous steroids are standard additional therapies. These will be allowed according to local practice, and randomisation will occur after plasma exchange. Patients in the cyclophosphamide limb will receive azathioprine as remission maintenance therapy. Patients receiving rituximab will not receive any additional maintenance therapy.


1.3. Rituximab

Rituximab is an anti CD 20 chimeric mouse/human monoclonal antibody, with human IgG1 constant regions and murine light/heavy chain variable regions (7). CD 20 is a ligand which exists on developing B cells, excluding stem cells and plasma cells. The role of the CD 20 ligand in nature is not fully understood, although mediation of apoptosis has been suggested. The mechanisms by which rituximab causes B cell depletion may involve complement induced B cell lysis, Fc receptor mediated cytotoxic cell killing or the direct induction of B cell apoptosis by rituximab. Rituximab therapy correlates positively with B cell depletion and rituximab levels. Different dosing regimens have been trialled. In patients with systemic lupus erythematosus (SLE), the efficacy of rituximab is dependant upon B cell depletion. 4 infusions of 375mg/m2 rituximab infusions (one per week for four weeks) were required for this to occur (13,14). In 3 published reports 375mg/m2 has resulted in B cell depletion and clinical response in patients with refractory vasculitis (10-13). On the basis of these results a dose of 4 x 375mg/m2 infusions will be used in RITUXVAS. In RITUXVAS, 2 doses cyclophosphamide will still be administered with the 1st and 3rd rituximab infusions, for two reasons. Firstly, the necrotising cresentic glomerulonephritis associated with AASV progresses rapidly and the therapeutic effect of rituximab is delayed. The use of cyclophosphamide/high dose steroid, which are standard components of induction therapy, will allow adequate immunosuppression in the early crucial treatment of AASV. Secondly, human antichimeric antibody (HACA) formation has been reported in NHL, SLE, and RA with varying frequencies, (4.3% of patients in RA (8)). The long term implications of these antibodies are not known. However, the possibility of anaphylactic reactions and rituximab resistance with further treatments exists. Co-administration of an immunosuppressant effective in the treatment of vasculitis is thus a logical approach to minimise HACA development. Rituximab has now been used to treat 300,000 patients with NHL. Long-term safety regarding carcinogenicity and fertility has yet to be established. However, no major long-term adverse sequelae have been reported (1). Up to 50% of patients receiving rituximab for an indication will develop infusion reactions with symptoms including; fevers, chills, rigors, flushing, throat irritation RITUXVAS EUVAS Trial 9
EUDRACT 2005-003610-15 rash rhinitis fatigue headache, nausea, vomiting, urticaria, angioedema, bronchospasm, myalgia, arthralgia, hypotension, hypertension and exacerbation of angina or congestive cardiac failure. Later reactions include diarrhoea, leucopenia, neutropenia, thrombocytopenia, anaemia, and infections in 30%, which may or may not be drug related (investigators brochure).


1.4. Aims of RITUXVAS:

1 To assess the rates of preliminary response and sustained remission of AASV following rituximab (on the basis of former studies, 86% sustained remission expected with rituximab compared to 75% in control group). 2 To assess safety of a rituximab regimen in terms of severe adverse events (in patients receiving standard therapies, adverse advent rate is 45% at 2 years, at least 50% of which are infection relapted. In comparison rituximab use is only rarely associated with infections, therefore 22.5% adverse event rate expected with rituximab compared to 45% at 2 years in control group).


2. Hypothesis: Rituximab leads to a higher rate of sustained remission compared to standard therapies (cyclophosphamide/azathioprine) with a lower rate of severe adverse events and reduced cyclophosphamide exposure.


3. Trial Design

International, randomised, controlled, prospective, open trial comparing a rituximab based regimen with a standard cyclophosphamide/azathioprine regimen in the treatment of active ‘generalised’ AASV. Informed consent and ethics committee approval will be obtained.


3.1. Inclusion Criteria (all four must be present)

1. A new diagnosis of WG, MP or renal-limited vasculitis (RLV)(appendix 1) 2. Renal involvement attributable to active WG, MP or RLV with at least one of the following: a) Biopsy demonstrating necrotizing glomerulonephritis. b) Red cell casts on urine microscopy or ≥ ++ haematuria 3. ANCA positivity ANCA positivity requires either (a) or (b) (a) PR3-ANCA by ELISA or a typical cANCA pattern by indirect immunofluorescence (IIF), or both. (b) MPO-ANCA by ELISA. A positive pANCA by IIF requires confirmation by MPO-ANCA ELISA.



3.2. Exclusion Criteria

1. Previous cyclophosphamide, (greater than 2 weeks of an oral or IV pulse cyclophosphamide regimen). 2. Co-existence of another multisystem autoimmune disease, e.g. SLE, Churg Strauss Syndrome, Henoch Schonlein Purpura, rheumatoid vasculitis, essential mixed cryoglobulinaemia, anti-glomerular basement membrane antibody positivity 3. Hepatitis B e antigen positive or Hepatitis C antibody positive. 4. Known HIV positive (HIV testing will not be a requirement for this trial). 5. Previous malignancy (usually exclude unless agreed with trial co-ordinator). 6. Pregnancy, breast feeding or inadequate contraception if female. 7 Allergy to a study medication 8 Live Vaccine within last 4 weeks



3.3. Randomisation

1 Variables known to influence primary end points will be balanced between groups by minimisation i Age ii Disease WG or MP/RLV iii Creatinine 2 Randomisation form (see TMF) sent by e-mail rbjones@doctors.org.uk or fax to 01223 586506 (clinical trials office).

3.4. Endpoints Primary end points will be assessed upon trial completion at 2 years. However interim analyses will be performed when 1 30 patients have completed 6 weeks, to assess efficacy (treatment response) and safety (severe adverse events). 2 40 patients have completed 6 months to assess efficacy (remission rates) and safety (severe adverse events).

i Primary

1 Sustained remission (BVAS = 0 at 6 months and sustained for 6 months). 2 Severe adverse events (CTCAE grade ≥ 3) at 2 years.

ii Secondary 1 Efficacy

Response rate at 6 weeks (BVAS < 50% baseline) Remission at 6 months (BVAS=0 for 2 months by 6 months) Time to remission (BVAS=0) Relapses (all relapses and major/minor) BVAS area under the curve Change in GFR Change in SF-36 Change in VDI

2. Safety Severe adverse events (CTCAE grade ≥ 3) at 6 weeks and 6 months

All adverse events Death Prednisolone cumulative dose Cyclophosphamide cumulative dose

iii Tertiary

Human anti-chimeric antibody testing Correlation of B cells with disease activity Change in ANCA and disease activity Histopathology predictors of outcome



3.5. Interventions

I Drug regimens a) Rituximab Regimen: Rituximab, 375mg/m2 IV once a week for 4 weeks (i.e. 4 doses total), with 2 doses of cyclophosphamide 15mg/kg, 2 weeks apart given with the 1st and 3rd rituximab dose (appendix 3).

b) Control (cyclophosphamide/azathioprine) Regimen; Cyclophosphamide 15mg/kg for 3-6 months (6-10 doses total) to be given IV according to protocol (appendix 3) for remission induction. Cyclophosphamide should be converted to azathioprine for remission maintenance. c) Steroids. All patients will receive 1g IV methylprednisolone, then same daily oral corticosteroid regimen (appendix 3). d) Plasma exchange or IV methylprednisolone will be allowed according to local practice for patients with organ threatening disease. NB randomisation should not occur until completion of plasma exchange to avoid loss of rituximab during plasma exchange. The first dose of cyclophosphamide can be given prior to completion of plasma exchange. e) Progressive disease Within the first 6 months disease progression defined as a persistence of nephritic sediment or activity on a renal biopsy and a failure to improve GFR≥ 10 mls/min, if GFR at diagnosis is < 50 mls/min (calculated by cockcroft gault formula, see TMF for formula) OR persistence or new occurrence of a major non-renal BVAS item at 6 weeks then additional treatment should occur: i) Rituximab limb: 3rd dose of cyclophosphamide (15mg/kg) ii) Cyclophosphamide limb: Plasma exchange or IV methylprednisolone (according to local practice). f) Relapse Therapy. Relapses will be categorised as major or minor.

i) Rituximab limb: Rituximab with steroid will be used for major and minor relapse. Additional cyclophosphamide may also be used for major relapse.

ii) Control limb: Increased azathioprine and steroid for minor relapse and cyclophosphamide and steroid for major relapse.

II Evaluations Study assessments (including history and examination) will be performed at entry, 1.5, 3, 6, 9, 12, 15, 18, 21 and 24 months and at the time of relapse (see appendix 5, and TMF for assessment schedule). At each assessment the following should be performed: a) Blood for routine testing. b) 10 mls serum saved for centralised immunological analysis (including ANCA) and HACA. c) BVAS for each assessment and at relapse;

Every 6 months SF-36 and VDI should be performed (23-25)(see TMF).

Initial Renal histology will be reviewed by a EUVAS panel of histopathologists, based in Milan.



3.6. Withdrawal and treatment failure

1 At patient's or physician's request. Reason for withdrawal is to be recorded in the CRF. 2 Patients not achieving remission within 6 months to be withdrawn from trial drug regimen and will be treated according to local practice. They will remain under trial follow up.


3.7. Adverse events

1 Adverse effects will be actively sought and recorded in CRFs at each evaluation (see section 4.5 and TMF). 2 Unexpected, severe adverse events attributable to study medication must be reported within 24 hours to trial management committee. (see TMF and section 4.5 for full details).


3.8. Statistical analysis

Primary end points will be assessed upon trial completion at 2 years. However interim analyses will be performed: (a) When 30 patients have completed 6 weeks trial participation to assess (1) treatment response (BVAS fall of > 50% baseline). (2) safety (adverse events CTCAE grade 3 or greater) and (b) When 40 patients have completed 6 months trial participation to assess (1) remission (BVAS 0 for 2 months by 6 months) (2) safety (adverse events CTCAE grade 3 or greater)Primary end point confidence Intervals. A total of 30 patients will receive rituximab. The expected sustained remission rate for rituximab is 86%, based on the results of former studies. The 95% confidence interval for a sustained remission rate of 86% is 70.3%-94.7% when the sample size is 30. The expected response rate of 86% is considered clinically significant as patients with active newly diagnosed generalised AASV without 3-6 months of cyclophosphamide are conventionally expected to experience disease progression (worsening of clinical signs and symptoms). The expected sustained remission rate in the control group is 75%. Analyses on the 40 patients should be considered exploratory.


3.9. Duration: 3 years: 6 months recruitment, two year follow-up per patient and 6 month analyses.


Visit the EUVAS website for details.