Salivary biomarkers for primary Sjögren's syndrome detection. A multi-center study. (Biomarker development study)

Sjögren*s syndrome (SS) is a progressive, chronic autoimmune disease
characterized by sicca symptoms of oral (xerostomia) and ocular dryness
(keratoconjunctivitis sicca), which affects ~4 million Americans, mostly women
at a ratio of 9:1 with men. Primary SS (pSS) occurs alone while secondary SS
(sSS) presents in connection with other autoimmune diseases such as rheumatoid
arthritis (RA) or systemic lupus erythematosus (SLE). Five to 10% of pSS
patients develop B-cell lymphoma in their salivary glands, usually a mucosa
associated lymphoid tissue (MALT) lymphoma.
Current diagnosis of pSS is clinically challenging, as it relies on assessment
of criteria established by the 2002 American European Consensus Group (AECG)
that contains six components. Sicca patients (those with subjective dry eyes
and dry mouth) are evaluated using the six AECG criteria. All three clinical
centers for this study (UMN, OMRF and UMCG) adhere to the AECG criteria for pSS
diagnosis. These include: I) establishing ocular symptoms; II) establishing
oral symptoms; III) objective evaluation of ocular signs; IV) assessing the
histopathology of salivary gland specimens for patterns consistent with
Sjögren*s syndrome; V) objective evaluation of salivary gland involvement (such
as decreased salivary flow rate); and, VI) determining the presence of anti-SSA
and anti-SSB auto-antibodies in the serum. Based on the AECG criteria, pSS is
defined as 1) the presence of 4 of the 6 criteria, as long as either
histopathology of salivary gland specimen or serology for SSA and SSB
auto-antibodies is positive; or 2) the presence of any 3 of the 4 objective
criteria items (ocular signs, histopathology, salivary gland involvement, and
serology). It should be noted that this clinical research project focuses on
pSS and not secondary SS (sSS), as sSS patients are a less coherent cohort with
a large component of related autoimmune disorders that will affect salivary
biomarkers* behavior and profile.
In April of 2012, the American College of Rheumatology (ACR) gave provisional
approval to a new set of classification criteria for SS proposed by the
NIH-funded Sjögren*s International Collaborative Clinical Alliance (SICCA).
Under the new ACR criteria, SS case definition requires at least two of the
following three objective features: 1) positive serum anti-SSA or anti-SSB
antibody test or (positive rheumatoid factor and antinuclear antibody titer
>=1:320), 2) keratoconjunctivitis sicca (KCS) with ocular staining score >=3, 3)
presence of focal lymphocytic sialadenitis with a focus score >=1 focus/4 mm2 in
labial salivary gland biopsy samples. In their analyses, the SICCA expert
panel noted that the observed agreement with the AECG criteria was high when
all objective tests were available to define the AECG criteria. The SICCA
analyses, partly due to the inclusion criteria of participants of the SICCA
study, also found that subjective complaints lacked diagnostic specificity.
Therefore, for study purposes, we will perform primary data analyses of
biomarker association with SS diagnosis based on provisional ACR criteria.
However, since another professional group, the European League Against
Rheumatism (EULAR), is considering whether they either will endorse the new
SICCA classification criteria or will ask for modification of these criteria,
we will also analyze biomarker performance using the full AECG criteria set.
It is possible that future criteria endorsed by both the ACR and EULAR could
contain additional elements from the AECG criteria or that elements of the ACR
criteria set will be omitted or be replaced by elements from the AECG criteria.
Correct diagnosis and classification of SS patients continues to be a
substantial problem, sometimes taking 10 years or more. Early and accurate
diagnosis of SS is crucial as we look for ways to slow or halt the progressive
nature and glandular destruction. The goal of this study is to perform a
biomarker development study for salivary SS individual biomarker confirmation.
Using the biomarker development findings, a panel will be built and evaluated
against diagnosis based on ACR criteria. The final panel, if it achieves its
performance criterion, will be fixed for an independent biomarker validation
study. The overarching clinical goal of the study is to reduce the number of
unnecessary diagnostic workups for non-SS sicca patients. Based on our
preliminary study, the salivary biomarkers selected will have 98% sensitivity
and at least 50% specificity for SS detection. This will permit at least half
of the non-SS sicca patients to avoid the diagnostic workups. Our collective
clinical experience indicates that 30-50% of sicca patients will be diagnosed
with pSS based on the AECG criteria. The evaluation of salivary markers for pSS
detection in primary referral clinics, where clinical impact can be more
significant, may reduce the number of diagnostic workups and aid in early
detection of pSS.
The salivary biomarkers developed for SS detection in this research project
will be clinically impactful in the following ways:
1) Preliminary studies suggest the combination of salivary biomarkers can
achieve sensitivity of 98% and specificity of 50%, which could reduce
unnecessary diagnostic workups in approximately 50% of the non-SS sicca
patients. This will be clinically relevant considering over 60% of sicca
patients will not have a SS diagnosis.
2) Clinicians who suspect that a patient has SS first will do a saliva test to
see whether the patient*s biomarkers are consistent with SS. The intended use
will be as a screening/risk assessment test for a symptomatic, undiagnosed
population. If a marker is positive, the diagnostic evaluation for SS is
justified to evaluate, assess and treat the patient. If a patient does not
fulfill the diagnostic criteria for pSS, they may have components of the
disease spectrum (e.g., keratoconjunctivitis sicca (KCS), xerostomia, or
extraglandular manifestations or hyposalivation) that need treatment. When a
patient is considered for diagnostic work up and a physician/dentist is
familiar with the pattern of complaints related to SS, he or she can decide to
restrict the diagnostic workup to the saliva test when the patient's history is
not very characteristic for SS. This will reduce the number of diagnostic work
ups for SS, as the work-up will occur only if there is a strong suspicion that
the patient has SS.
3) Assessing the current 6 criteria for AECG diagnosis for pSS as well as the 3
criteria for ACR diagnosis is invasive, time consuming and costly. It often
requires months to receive serology and salivary gland biopsy (histopathology)
results. The total evaluation time is up to 12 weeks. A salivary pSS test could
be completed at a fraction of the time and costs.

The objectives of this research study are to perform a biomarker development
study for salivary pSS biomarker confirmation and panel building. The findings
will be analyzed against SS diagnosis based on ACR criteria:

Aim 1
To test individual biomarker association (OR) with SS diagnosis based on ACR
criteria and test an initial panel sensitivity and specificity for diagnosis of
SS at the time of interim analysis using paraffin stimulated whole saliva
specimens collected from the first 210 recruited subjects.

Aim 2
To confirm the sensitivity and specificity of the panel built from Aim 1,
refining the panel and evaluating its sensitivity and specificity on specimens
from all 420 subjects.

From 420 patients complaining of a dry mouth and dry eyes that are referred to
one of the three Sjögren's referral centers participating in this study for a
diagnostic work-up of Sjögren's syndrome, a whole saliva sample will be
collected at the first visit to one of these centers for their oral work-up.
These samples will be send to UCLA and analysed according to the UCLA protocol
(salivary biomarker test) to rate these samples as matching for Sjögren's
syndrome or not.. Separately from this analysis at UCLA, the diagnostic work-up
for Sjögren's syndrome will be completed (subjective dry mouth, subjective dry
eyes, saliva collection, eye analysis, salivary gland biopsy, serology) to jude
whether these patients fulfill the ACR criteria for Sjögren's syndrome.
Finally, the UCLA data will be matched with the outcome of the diagnostic
work-up for Sjögren's syndrome at the three centers. On basis of this analysis
it will be decided whether the newly developed diagnostic salivary biomarker
test match the results of the ACR diagnostic work-up in order to judge whethr
the salivary biomarker test has a sufficient high sensitivity and specificity
to replace (some of) the ACR diagnostic work-up tests. As as part of this study
also a full diagnostic work -up according to the AECG criteria will be done,
the various analyses will also be done for this criteria set.

Saliva is without any burden and risk for the patient. However, as for the ACR
criteria yet a labial biopsy is required for diagnosing SS and is has been
reported in the literature that there is a slight risk that havesting labial
salivary glands can result in some numbness of the biopsy area in the lip,
there is an inherent risk of inducing some numbness of the lip in a minority of
the patients. It has to be noted that taking a labial biopsy is routine care in
all Sjögren's centers worldwide taking the slight risk of inducing numbness of
an area of the lip into account. This risk is existent, although very small,
when a proper technique is used.