Clinical application of gene expression analysis in patients with insect venom allergy

Insect venom allergy (defined as at least one life systemic IgE mediated
reaction in lifetime after an insect sting) is present in approximately 1-3% of
population [1].
The treatment of choice in insect venom allergy (IVA) patients is venom
immunotherapy (VIT) with bee, wasp or Polistes venom. VIT has two phases: (1) a
built-up phase where increasing amounts are administered until the maintenance
dose of allergen has been reached and (2) a maintenance phase where the
maintenance dose is administered every 4 to 6 weeks. The built-up phase lasts
from 1 day to 7 weeks depending on the protocol. In the UMCG patients start at
the day care reaching 1/10th of the maintenance dose, and continue in the
outpatient clinic with weekly one injection with increasing amount of venom
during about 6 weeks. At this time the risk at a systemic reaction to a
subsequent sting is reduced from 70% (before the start of VIT) to 3% after
reaching maintenance dose [5]. To reach long-term protection the maintenance
phase has to be continued during at least 3 years (in patients with mild
systemic reaction, grade I-III Mueller) to 5 years (in patients with
anaphylaxis (grade IV Mueller), enabling the lifelong prevention of
anaphylactic reactions even after stopping VIT in most patients.
After stopping VIT the risk at a re-systemic reaction increases in some
patients, in most patients it remains low. Unfortunately it is not possible to
predict inefficacy in the individual patient. Some parameters are of influence.
Firstly, the duration of treatment influences the risk at a re-systemic
reaction: after 2 years of VIT the risk is higher than in patients where VIT
has been stopped after 3- 5 years maintenance course (30% vs 3%) [1,5].
Secondly it is known that patients with side effects during treatment are more
prone for inefficacy; prolongation of VIT is able to reduce the risk at a
re-systemic reaction [1,5]. Thirdly, the amount of allergen might be not
enough. It has been shown that continuation of VIT with an increasing dose (eg
200 ug) is able to reduce the risk in these patients [9]. Fourthly, it is known
that it depends on the culprit insect: in wasp venom allergic patients: the
long-term effectiveness of therapy is assessed as 85-95%, but in patients
allergic to bee venom it is about 75-85% [1]. Fifthly, the severity of the
sting reaction: the less severe, the better the protection. Overall it is
known that 10-20% of subjects remain vulnerable to insect venom in spite of
completion the treatment [2]. Unfortunately it is not possible to predict
inefficacy of treatment so far [3,4,5,6,7].
The immunological mechanism(s) responsible for long-term protection
achieved in VIT are mainly unknown [7]. It is hypothesized that it is related
to mast-cell inactivation [7], a shift from Th2 to Th1 cytokine induction [8],
lymphocyte Treg up regulation and T cell tolerance induction [8], monocytes
activation [3] and/or suppressed antigen presentation by dendritic cells [8].
It seems quite likely that more than one pathway and more than one cell type
are involved and responsible for the effect of VIT. Thus gene expression will
be studied in peripheral blood using mRNA from all cell types without cell
separation. Additionally the protocol of RNA isolation and gene expression
analysis used in the protocol are standardized methods which avoid human
laboratory errors and might by adapted to clinical practice in the future.
Differences in gene expression will be studied in different patient groups: in
group 1 (the long term protection after VIT was achieved * finished VIT, re
stung without reaction) and 2 (the immunological mechanism responsible for
protection was not effective* finished VIT, re stung with anaphylaxis) will be
related to the gene expression in group 3 and 4 (resp. highest (before VIT) and
lowest (3 to 5 years of VIT) -risk of systemic reaction after resting). The
gene expression profile achieved after 3-5 years of VIT might be similar to the
gene expression profile in patients who do not react after re-sting, whereas
gene expression profile of those who react in spite of treatment might be
comparable with untreated patients.
Better understanding of molecular differences in patients with venom allergy
after completion of VIT may elucidate molecular modifications in pathways that
predict ineffective treatment. This will enable to predict the necessity of
modification of treatment in individual patients. The treatment may be modified
by (1) prolongation the treatment period or (2) increasing the dose of drug to
improve the protection against anaphylaxis. The results of the project may also
allow for creating new diagnostic methods of insect venom allergy.
If we find clear cut differences in the patient group we will seek to confirm
this result in a larger group of patients in cooperation with the insect venom
allergy interest group of European Academy of Allergy and Clinical Immunology.

Primary objective:

Is there a gene expression profile which may predict the long term effect of
venom immunotherapy?

Secondary objective:

Which genes are differentially expressed after venom immunotherapy?

Description This is an observational case-control study with a retrospective
analysis of the follow up of the patients treated with VIT to evaluate to the
long-term protection in patients who experienced a re-sting, comparing
1- those without a reaction at a re-sting after stopping VIT
2- to those who did experience a re-systemic reaction at a re-sting after
stopping VIT
and subsequently compare the gene expression profile of these patients.
Patients will be recruited from the insect venom allergy database of Department
of Allergology. .The patients will be asked if they are willing to visit the
outpatient department and take part in the study.
In addition, two groups of patients will be studied to relate the results to
the changes in gene expression during VIT:
3 - patients who routinely start VIT at the day care of allergology of UMCG
4- patients who routinely visit the outpatient department of allergology
of UMCG after 3-5 years of VIT
The differences in gene expression profiles will be studied. Those subjects who
react to a re-sting in spite of treatment with VIT probably have lost
protection or never had protection induced by immunotherapy. In patients before
VIT the risk at a systemic reaction when restung is about 70%, whereas in
patients who completed VIT this risk is reduced to about 3% [1,5]. Thus the
results in groups 3 and 4 are essential to understand the importance and
findings in groups 1 and 2.

Duration of the study: 2008-2009

Procedures
Patients of group 1 and 2 will be asked to visit the outpatient clinic in order
to collect blood samples (see above).
The samples in the group 3 will be taken just before the start of VIT in the
hospital at the daycare. In the group 4 blood samples will be collected when
routinely visiting the outpatient clinic after 3 to 5 years of VIT.

The proposed study has no risks for the health of investigated individuals.
The burden for patients in group 1 and 2 is related to the unscheduled visit in
the outpatient department; therefore travel cost compensation is planned.
The burden for patients in group 3 and 4 is minimal because the blood samples
will be taken as a routine procedure.
The benefit for patients is different with respect to the different patient
groups. Subjects with a reaction after re-sting may prolong the immunotherapy
or increase the dose of allergen used to gain better protection from systemic
reactions after resting.
Remaining subjects will be informed about the results of the gene expression
profile.
The results of the study might be important for the assessment of the risk of
insect sting anaphylaxis and to advice on preventive methods to the individual
patients.