Discussion
Peanut allergy is one of the most frequent causes of severe
food-associated anaphylaxis. To prevent or minimize such systemic,
potentially life-threatening allergic reactions, accurate and reliable
diagnostic tools that are easy to implement and safe are urgently
required. Moreover, diagnostic accuracy and the reliability of negative
values in sensitized individuals is needed to avoid unnecessary
diagnosis with all the consequences that come with the diagnosis of
peanut allergy. In this study, we sought to investigate the clinical
utility of the recently developed in vitro Hoxb8 MAT for the
diagnosis of peanut allergies and to compare it to established and
validated clinical diagnostic tests including SPT, sIgE measurements and
BAT. For this purpose, we analyzed serum samples that have been
prospectively collected in the context of MONAS and were subsequently
stored long term in a frozen state (-80°C) in a blinded manner.
Importantly, the BAT has previously shown high accuracy in the diagnosis
of peanut allergy in multiple studies . Our results show here strongly
suggest that the Hoxb8 MAT performs as well as the BAT in terms of
diagnostic accuracy in a mixed cohort of severe and clear peanut
allergic patients and those with an equivocal history. The respective
test is at the same time more accurate as commonly performed tests like
the SPT and sIgE measurement.
While the idea to perform MAT for diagnostic purposes is not new , its
broader implementation has been hampered by scalability limitations. In
one study, Bahri et al. generated mature mast cells from isolated human
primary blood stem cells. Their results concerning diagnostic accuracy
were similar to those we present here using Hoxb8 MCs. However, their
assay relied on a sophisticated mast cell differentiation protocol,
which required 8-10 weeks to generate a limited number of cells
available for the diagnostic assay. Another challenge of this approach
is the variability between different blood stem cell donors, which might
affect assay robustness and diagnostic reproducibility, unless cells of
various donors are pooled to minimize variability each time. These
issues have been resolved with the Hoxb8 MC system, as mature mast cells
can be repetitively differentiated from the same immortalized mast cell
progenitor stock at unlimited numbers within only 5 days. In another
study, Santos et al. have used the human LAD2 mast cell line for the
diagnosis of peanut allergies . The result also looked encouraging.
However, LAD2 cells generally feature slow growth characteristics with a
doubling time of 2 weeks . Hoxb8 MC progenitors double roughly once
every 24 hours .
Hoxb8 MCs showed optimal cut-off values ≤5.2%, which is comparable to
the cut offs reported for BAT , as well as maximal activation of
>89% in our diagnostic performance study. These findings
suggest that the Hoxb8 MAT overcomes prior limitations of other MAT
approaches while maintaining excellent diagnostic accuracy.
From a methodological standpoint the BAT and the MAT both represent
similar cell-based functional in vitro tests, which rely on flow
cytometric identification of activated allergic effector cells upon
allergen stimulation. However, there are major differences between these
two assays. The BAT is performed with patient’s whole blood, which must
be processed and measured within maximally 24 hours to ensure valid
results . The MAT, on the other hand, relies on serum samples that can
be stored in the freezer over months (even years) and analyzed in large
batches. This is particularly important for large studies or
multi-center clinical trials as the analysis of serum samples can be
done retrospectively in a centralized manner. The serum samples included
in this study were analyzed in just 2 days. Thus, the Hoxb8 MAT clearly
has the potential to overcome logistical challenges that have hampered a
broader implementation of the BAT in clinics so far.
Direct comparison of activation results between the BAT and the Hoxb8
MAT revealed that the BAT requires roughly 100-times lower allergen
concentrations to induce comparable percentage of cell activation. One
potential explanation for this difference is linked to the number of
allergic effector cells present per sample. There are an estimated 0.5 x
106 leukocytes in 50 µl human whole blood , which is
the amount used for the BAT. Basophils make up roughly 0.1-0.5% of
leukocytes , which means that there are 500-2500 basophils present in
one BAT sample. In comparison, we were using 50 x 104mast cells per condition for the Hoxb8 MAT. Thus, there were 20 to
100-times more cells present in the Hoxb8 MAT than in the BAT, which
could explain the observed difference in allergen concentration needed
to reach an equivalent percentage of activated cells. However, this
hypothesis has to be further investigated.
Despite losing intrinsic cellular information (i.e. patient’s own versus
lab grown allergic effector cells), the Hoxb8 MAT showed comparable
diagnostic accuracy as the BAT in this study, further supporting the
notion that humoral components contained in the serum are sufficiently
representative of a patient’s allergy status. Furthermore, in
approximately 10% of cases the BAT results in false negative or false
positive outcomes with basophils that do not show any activation upon
FcεRI-mediated stimulation (non-responders) or with unspecific
activation of basophils in the negative control . It has been
demonstrated that basophils of so-called non-responders feature an
impaired signaling cascade with low tyrosine kinase Syk levels . These
individuals pose an important diagnostic challenge in the daily clinical
allergy workup. It is also important to note, that many diagnostic
performance studies using BAT exclude these non-responder samples and
thereby do not entirely represent the real-life situation . As the
initial dataset we used in this study did not contain non-responder
samples, the diagnostic accuracy of the BAT in our ROC curve analysis
might be overestimated as in many other studies. Importantly, we
provided strong evidence in a separate experiment showing that the Hoxb8
MAT is able of accurately capturing these non-responder samples and
might thereby overcomes this current diagnostic issue .
Overall, this study has demonstrated that the Hoxb8 MAT features
excellent diagnostic accuracy and outperformed both SPT and sIgE
measurements in the diagnosis of peanut allergy. While it represents a
valuable alternative to established allergy diagnosis tests further
studies including other allergens and allergen components will be
conducted in near future.
References
Acknowledgements: We thank all the people from the Kaufmann,
Eggel and Eiwegger laboratories. We also acknowledge members of ATANIS
Biotech AG, Prof. Dr. med. Jean-Pierre Kinet, Charlène Niogret, Marianne
Zwicker and especially Antonia Ferreira and Sophie Buchser for valuable
scientific discussions and support with experimental procedures and
protocols.
Competing interests : NBZ is a co-founder, shareholder, and
employee of ATANIS Biotech AG. AC has nothing to declare. RvB is an
employee of ATANIS Biotech AG. JAH has nothing to disclose. XY
has nothing to disclose. CMD has nothing to disclose. LD has nothing to
disclose. JEMU reports grants and personal fees from ALK-Abelló A/S,
personal fees from Bausch Health, personal fees from Kaleo, grants from
DBV Technologies, grants from Regeneron, and grants from Food Allergy
Anaphylaxis Programme (SickKids), outside the submitted work; and
Section Chair of Food Allergy and Anaphylaxis, Canadian Society of
Allergy and Clinical Immunology; Healthcare Advisory Board, Food Allergy
Canada. TK is a co-founder, shareholder and board member of ATANIS
Biotech AG. AE is a co-founder, consultant and shareholder of ATANIS
Biotech AG. AE received grants from ATANIS Biotech AG. TE reports grants
from CIHR, FWF, Food Allergy and Anaphylaxis Program Sickkids, ALK. He
is site PI of company sponsored trials by DBV, Novartis and Stallergenes
Greer, EFSA and FARE. Personal fees from Danone/Nutricia/Milupa,
ThermoFisher, Aimmune, Stallergenes Greer, ALK, MADX and Non-financial
support from Novartis and MADX, all outside the submitted work. He is an
associate editor of Allergy.
Author contribution: NZ and RvB performed Hoxb8 MAT and did
data entry. AC analyzed data and critically reviewed the manuscript.
JAH, XY and CMD performed BAT and did data entry. LD cross checked
clinical accuracy and contributed to clinical data entry. JEMU cross
checked clinical accuracy and contributed to clinical data entry,
contributed to recruitment and contributed to manuscript revision. TK
contributed to Hoxb8 MC generation, provided advise on study design and
critically reviewed the manuscript. AE and TE co-designed the study,
co-contributed to generation of the manuscript, and co-supervised the
project and collaboration. All authors reviewed the results and
commented on the manuscript.