Precision Medicine in Lups Nephritis: A Multicenter Proof-of-concept Study for Histopathological Biomarkers Analysis in Renal Biopsy
Conditions: Lupus Nephritis, Systemic Lupus Erythematosus
Sponsor: Fundacin Biomedica Galicia Sur

“PRECISION MEDICINE IN LUPUS NEPHRITIS: A MULTICENTER PROOF-
OF-CONCEPT STUDY FOR HISTOPATHOLOGICAL BIOMARKERS
ANALYSIS IN RENAL BIOPSY”
Unique Protocol Identification Number  ISS 13844
DOCUMENT DATE: 18TH OCTOBER 2021

INTRODUCTION
Lupus nephritis (LN) may affect approximately half of patients with Systemic Lupus
Erythematosus  (SLE). LN  is  a major cause of morbidity  and the most important
predictor of mortality in patients with SLE (1,2). Some 5-20% of patients with LN may
develop end-stage renal disease within 10 years of follow-up from the time of diagnosis
(3). Other studies have described progression to end-stage renal disease in 10-30% of
patients with LN (4,5).  
The European League Against Rheumatism (EULAR), the European Renal Association
(ERA) and the European Dialysis and Transplant Association (EDTA) have recently
updated their recommendations for the management of LN. These recommend the use
of  intravenous  (IV)  methylprednisolone  boluses  followed  by  lower  doses  of  oral
glucocorticoids  (GC)  and  place  mycophenolate  mofetil  (MMF)  and  the  European
regimen of cyclophosphamide (CYC) as the immunosuppressive drugs of first choice,
with the IV CYC regimen for certain more aggressive cases. They also consider the use
of "multitarget therapy" based on the combination of tacrolimus (TAC) and MMF and
GC in patients with proteinuria in the nephrotic range who have not responded to the
first line of treatment. For refractory active renal disease, they recommend as an
alternative the use of rituximab (RTX) 1000 mg IV repeated after 15 days (6). 
Recently, belimumab has been shown to be significantly more effective than placebo in
the treatment of patients with active LN (7). This finding will lead to positioning
belimumab in the therapeutic algorithm for LN.  
However, in clinical practice these immunosuppressive drugs are not always effective in
the treatment of LN, and even one in 3 patients with an initial favorable response may
experience renal recurrence (3). 
The choice of the appropriate treatment for LN and its early initiation are key to
improve the prognosis of these patients and to avoid progression to chronic renal
failure.
The identification of biomarkers capable of predicting the response (or lack thereof) to
one or another therapy at the time of LN diagnosis would allow to implement precision
medicine, thus constituting a revolution in the treatment of patients with LN. Being able
to make personalized therapeutic decisions, based on the biological characteristics of
each patient's renal sample, allows more targeted treatments with greater specificity to
be established. 
The  objective of this project is to analyze histopathological biomarkers in the renal
biopsy to predict the renal response to the different drugs used in the treatment of LN.
This would contribute to a more specific and cost-effective therapeutic strategy.  
PATIENTS AND METHODS
Inclusion criteria:
Patients  with  a  diagnosis  of  SLE  according  to  the  1997  American  College  of
Rheumatology (ACR) criteria (8).  

Diagnosis of lupus nephritis type 3,4,5 or mixed forms of the above according to the
International Society of Nephrology (ISN)/Renal Pathology Society (RPS) classification
of LN (9). 
Date of performance of the renal biopsy: year 2000 or later. The reason for this time
frame is the theoretical homogenization in the therapeutic management of LN with the
introduction of MMF into the therapeutic armamentarium of LN and the extension of
the use of the European intravenous cyclophosphamide guideline.
Availability of the patient's first renal biopsy specimen preserved for re-evaluation.
Availability of essential clinical data to carry out the study.
Signature of the informed consent by the patient.
Exclusion criteria:
Refusal by the patient to sign the informed consent.
Sample size
For this proof-of-concept study, our objective is to analyze around  60 renal biopsy
samples (expandable according to the results obtained). 
Methods:
The following general variables will be collected: 
- demographic data: age, sex, ethnicity.
- clinical data on SLE:
- diagnosis and chronological data: time of SLE diagnosis, time of LN diagnosis,
time form SLE diagnosis to LN.
- 1997 ACR SLE classification criteria,
- activity: involvement of different organs and systems by SELENA-SLEDAI 
(10) at the time of LN diagnosis and at the time of the last evaluation of the 
patient (or death, if applicable).
- damage: by item and by domain according to the Systemic Lupus International 
Collaborating Clinics Damage Index (SDI) (11) at the time of LN diagnosis and
at the time of the last evaluation of the patient (or death, if applicable).
- comorbidity: arterial hypertension, diabetes, dyslipemia, smoking habit, severe 
infections, neoplasms, etc. at the time of LN diagnosis and at the time of the last 
evaluation of the patient (or death, if applicable).
- laboratory data at the time of LN diagnosis and at the time of the last evaluation of the
patient (or death, if applicable):
- blood (general tests): acute phase reactants (erythrocyte sedimentation rate and 
C-reactive protein), full blood count, creatinin, glomerular filtration rate, blood
urea nitrogen, liver function tests, lipid profile.

- blood (serological tests): complement (C3 and C4), levels of anti-dsDNA  
antibodies, antiphospholipid antibodies: anticardiolipin (Ig M and Ig G), anti-
beta2GP-1 (Ig M and Ig G), lupus anticoagulant.
- urine: hematuria, piuria, proteinuria, casts.
- histopathological markers of renal biopsy:
- histological class according to the 2003 ISN/RPS classification (9). 
- National Institutes of Health (NIH) activity index (12): score (maximum 24)
and by item (endocapillary hypercellularity, neutrophils/karyiorrhexis, hyaline 
deposits/wire  loops,  fibrinoid  necrosis,  cellular  o  fibrocellular  crescents,  
interstitial inflammation).
- National Institutes of Health (NIH) chronicity index (12): score (maximum 12)
and  by  item  (global  glomerulosclerosis,  fibrous  crescents,  tubular  atrophy,
interstitial fibrosis).
- markers related to B lymphocytes  which may include but is not limited to:
CD19, CD20 and CD138.
-  markers  related  to  BLyS  (B  lymphocyte  stimulator)  and  its  functional  
consequences which may include but is not limited to expression of BLyS and
its receptors: BAFF-R, BCMA and TACI.
- markers related to other cell lineages which may include but is not limited to:
CD3 for T cells and CD68 for macrophages.
- markers whose determination in urine has proved useful in the diagnosis and 
follow-up of LN which may include but is not limited to the proinflammatory
cytokine  Monocyte  Chemoattractant  Protein-1  (MCP-1)  and  the  Neutrophil
Gelatinase Associated Lipocalin (NGAL). 
- SLE therapeutical data (including duration of the treatment):
- treatments for SLE prior to the diagnosis of LN: antimalarials, glucocorticoids 
(maximum  dose),  immunosuppressants:  azathioprine  (AZA),  mophetil  
mycophenolate  (MMF),  cyclophosphamide  (CYC);  biological  therapies:  
belimumab, rituximab.
- treatment of LN: glucocorticoids (maximum  dose),  immunosuppressants:  
AZA, MMF, CYC; biological therapies: belimumab, rituximab.  
- therapeutical data of comorbidities (including duration of the treatment):  
antihypertensive agents, oral antidiabetics, insulin, hypolipidemic drugs. 
- Evolution/prognosis of SLE: accumulated damage by using SDI (11) (by item and by
domain), comorbidities accrual, severe infections, organ failure, death. 
Variables will be collected to establish different patterns of response to treatment
and evolution of LN: 

a)  complete  renal  response,  defined  according  to  EULAR/ERA-EDTA
recommendations (13): proteinuria <0.5 g/24 hours and (near) normal estimated GFR. 
b) partial renal response, defined according to EULAR/ERA-EDTA recommendations
(13): ≥50% proteinuria reduction to subnephrotic levels and (near) normal eGFR
c) no response: all the other cases.
d) proteinuria levels at 12 months of treatment,
e) renal relapse as defined as reproducible increase in uPCR to >1 g if the baseline value
was <0.2 g, to >2 g if the baseline value was between 0.2 g and 1 g, or more than twice
the value at  baseline if the baseline value was >1 g AND/OR reproducible decrease in
GFR of >20%, accompanied by proteinuria (>1 g), and/or RBC and/or WBC cellular
casts (yes/no and number of flares), e) time to first flare, f) chronic renal failure,
f) end-stage renal disease requiring dialysis and/or renal transplantation.
Data collection will be based on the clinical history of the patients included in the study.
Confidentiality will be respected in accordance with RD 1720/2007 and the Data
Protection Laws. Approval will be requested from the  Galician Clinical Research
Ethics Committee (CEIC) as well as from the CEIC of each center, if necessary. 
EXPERIMENTAL STRATEGY AND RATIONALE
In this project we will use renal biopsies from patients with LN. These samples,
preserved in 10% formaldehyde and embedded in paraffin blocks by the corresponding
Anatomic Pathology Services, correspond to patients with LN who underwent renal
biopsy in the centers participating in the project.
BLyS (B lymphocyte stimulator) plays a key role in the pathophysiology of LN (14).
Therefore, in this study we will focus on markers related to BLyS and its functional
consequences. On the one hand, we will analyze among others the expression levels of
BLyS and its receptors (BAFF-R, BCMA and TACI), since the expression levels of
BLyS and its receptors are elevated in serum and renal biopsies of patients with LN and
are associated with disease progression and severity (15-17). 
On the other hand, because BLyS induces B cell survival (14), we will analyze the
expression levels of different B cell markers such as CD19 and CD20 among others.
Finally, we will analyze the plasma cell marker CD138, since plasma cell infiltration is
associated  with  increased  severity  of  lupus  nephritis  (18).  We  will  analyze  the
expression levels of markers of other cell lineages such as CD3 for T cells and CD68 for
macrophages. 
We will also analyze at least 2 biomarkers whose urinary levels in patients with LN
have been associated with a worse prognosis of LN: the chemokine MCP-1 (monocyte
chemoattractant protein-1) and the enzyme NGAL (neutrophil gelatinase-associated
lipocalin) (19).

The expression of these markers will be initially determined by immunohistochemical
staining of renal biopsies, which are preserved in formaldehyde. The samples will be
stained and analyzed mainly in the Anatomic Pathology Service of the “Complejo
Hospitalario  Universitario  de  Vigo”  and  in  the  laboratories  of  the  “Instituto  de
Investigación Sanitaria Galicia Sur” (IISGS), although the different participating centers
will participate in this work to the extent possible and cost-effective for the project.
Different methods of quantification will be used in function of the stains of the different
markers.
STATISTICAL ANALYSIS
In the descriptive study, numerical variables will be expressed as mean ± standard
deviation (SD) or median and interquartile range (IQR), depending on whether the
distribution is normal or not, respectively. We will establish 2 groups of patients,
according to the response to treatment and the clinical evolution of the patient with LN
(i.e., complete remission yes/no). To establish differences between patients in these 2
groups, we will use the χ2 test for categorical variables or Fisher's exact test when the
expected frequencies are small, the t-Student test for normal continuous variables, and
the Mann-Whitney U test for variables with non-normal distribution.
Different methods to analyze and/or mitigate the missing data problem will be used. 
The percentage of positive cells and staining intensity will be correlated with the
different patterns of response to treatment and evolution of LN.
Univariate and multivariate linear logistic regression analyses will be performed to
explore  the  relationships  between  the  different  variables  studied  (clinical,
histopathological,  therapeutic...)  and  the  presence  of  the  different  renal  outcomes
(dependent variable). Values of p <0.05 will be considered significant.
We will carry out other different statistical methods according to the results that we
observe from our initial analysis.
Statistical analyses will be performed by the Statistical Specialist of the IRIDIS Group.  
TIMELINES
Development of the definitive protocol: month 1. 
Submission and Approval by Ethics Committee: months 1-3.
Renal samples identification and preparation: months 4-5.
Informed consent signatures: months 4-6.
Shipment of renal samples: months 4-6.
Anatomo-pathological and laboratory studies: months 7-12.
Review of clinical charts: months 7-12.

Monitoring of the database: months 13-15.
Statistical analysis: month 16.
Elaboration of the final report: month 17-18.
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10.1016/j.jtauto.2020.100042. PMID: 32743523; PMCID: PMC7388339.