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\begin{document}

\title{Circulating 25-hydroxyvitamin D in secondary hemophagocytic
lymphohistiocytosis}



\author[1]{Jiayu Huang}%
\author[1]{Wanying Cheng}%
\author[1]{Changfeng Man}%
\author[1]{Shengen Liao}%
\author[1]{Hongxia Qiu}%
\affil[1]{Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare immune
disease, accompanied by high mortality. Vitamin D (VD), as a marker of
inflammation, plays an important role in the regulation of the immune
system by binding to receptors of immune cells (T and B cells). The
purpose of this study was to investigate the expression level of vitamin
D in HLH and to clarify its relationship with prognosis. Method: We
conducted a retrospective analysis of 54 adult sHLH cases from November
2015 to December 2020. Results: Among the cohort, 49 (90.7\%) patients
with sHLH had VD insufficiency (25-hydroxy vitamin D {[}25-(OH)D{]}
\textless{}50nmol/L) and median level was 23.95 (range: 7.5-90.8)
nmol/L. Classified by 30nmol/L as the threshold value, there were no
significant differences on baseline characteristics between two groups,
except triglycerides (TG). Level of 25-(OH)D was significantly
associated with TG (r=-0.34, P=0.011) and ferritin (r=-0.26, P=0.055).
Patients with lower 25-(OH)-D level acquired shorter overall survival
(28 vs. 125 days P=0.041). After multivariable adjustment, 25-(OH)-D was
identified as an independent factor of survival (HR 0.364;95\% CI
0.183-0.724; P=0.004). A significant interaction was discovered at
25-(OH)-D with ferritin (P=0.009) through subgroup analysis. Vitamin D
is more likely to predict survival in elderly, male, MHLH, EBV
infection, hemoglobin\textless{}90g/L, platelet\textless{}100\selectlanguage{ngerman}×109/L,
fibrinogen\textless{}1.5g/L, triglyceride, ferritin
\textgreater{}10000ug/l, sCD25\textgreater{}20000ng/l group.
Conclusions: Patients with sHLH almost had vitamin D insufficiency and
vitamin D is an important prognostic protective factor for sHLH.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

Hemophagocytic lymphohistiocytosis (HLH) comprises a heterogeneous
spectrum of systemic disorder that are congenital (primary) or acquired
(secondary), with the most devastating characteristics and mortality
rate more than 50\% even with specific treatment\textsuperscript{1}.
Regardless of specific type, knowledge of the pathogenesis remains
deficient. There is a well-established association between immune
dysregulation and the development of HLH. Hyperplasia of
mononuclear/macrophagocytic system, persistent activation of
CD8\textsuperscript{+} T cells and dis-regulated function of natural
killer (NK) cells caused by various triggers, leading to uncontrolled
over-secretion of cytokines including interferon gamma (IFN-\selectlanguage{greek}γ\selectlanguage{english}), tumor
necrosis factor \selectlanguage{greek}α\selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}), interleukin 6 (IL-6), IL-10, and
macrophage-colony-stimulating factor (M-CSF), is widely believed to play
a predominant part\textsuperscript{2-6}. Therefore, it's of part
interest of realizing early diagnosis and judging prognosis, to initiate
the most appropriate treatment. Emerging researches showed that low
platelet, elevated serum lactic dehydrogenase, higher
lymphocyte/monocyte ratio could predict poor survival of HLH
patients\textsuperscript{7,8}. However, a common limitation of these
indicators in improving the overall picture is the inability to be
intervened directly. Then, we attempted to explore an index of
prognostic importance and therapeutic possiblity.

Vitamin D (VD) is one of the indispensable steroid hormones, with
well-documented function of maintaining the metabolism balance of human
calcium and the formation of bone\textsuperscript{9}. In addition,
awareness is growing of the role of VD in immune system optimization on
the basis of extrarenal activation by
monocytes/macrophages\textsuperscript{10}. Combined with receptor
expressed on immune cells (B cells, T cells and antigen presenting
cells), VD is associated with increased autoimmunity as well as
susceptibility to infection. With a long serum half-life, indistinctive
regulation of liver production and dependent on substrate concentration,
serum 25-hydroxyvitamin D (25(OH)D) provides the single best assessment
of VD status\textsuperscript{11}. Much recent works had demonstrated
that VD deficiency was closely related to various autoimmune diseases,
such as rheumatoid arthritis, systemic lupus
erythematosus\textsuperscript{12,13.} Also, suboptimal VD levels have
been demonstrated to be a significant independent risk factor in chronic
graft versus host disease and a promising prognostic predictor in
various lymphoma\textsuperscript{14,15}. So far, little research has
described the characteristic findings of serum VD in patients with HLH.
Therefore, the regulating activity of VD in immune processes-key
components of HLH pathogenesis- prompted us to explore this role, on
this account to extended treatment strategy and even improve overall
survival of HLH patients. With our known etiology, we hypothesized that,
for secondary HLH (sHLH) patients, vitamin D might possess a prognostic
importance and might have the ability in distinguishing the primary
triggers.

\textbf{Materials and Methods}

\textbf{Study patients}

A total of 54 adult cases with newly diagnosed sHLH at the First
Affiliated Hospital of Nanjing Medical University from November 2015 to
December 2020 was enrolled. All the diagnosis of sHLH was based on the
2004 HLH Diagnostic Criteria\textsuperscript{16}. The NK cells
cytotoxicity assay was not applicable due to the limitation of
technology. Excluded criteria included (1) history of rickets; (2)
history of parathyroid or metabolic diseases; (3) use of
over-the-counter or prescribed vitamin complements; (4) history of
severe liver and kidney dysfunction.

\textbf{Data collection}

Baseline patients' characteristics were collected on admission,
including age, gender, specific pathogenesis, Epstein-Barr virus (EBV),
complete blood cell count (CBC), fibrinogen (FIB), lactate dehydrogenase
(LDH), triglycerides (TG), albumin, calcium, \selectlanguage{greek}β\selectlanguage{english}2-microglobulin (\selectlanguage{greek}β\selectlanguage{english}2 -MG),
ferritin, serum soluble interleukin-2 receptor (sIL-2R, sCD25), fever,
splenomegaly and serum 25-(OH)D. Our previous studies have revealed
clinical significance of PET-CT in diagnosing lymphoma-associated HLH
(LHLH) \textsuperscript{17,18}. Therefore, in this study, lymphoma with
lymph node or bone marrow biopsy and suspected by PET/CT were regarded
as LHLH.

The evaluation of baseline vitamin D level was performed in the clinical
laboratory of our hospital and assayed by electrochemiluminescence
immunoassay (Rochee 170, Roche Co. Ltd. Shanghai, China). 25-(OH)-D
levels were categorized according to a cut-off value of a published
literature:30nmol/L\textsuperscript{19}.

\textbf{Follow-up}

Overall survival was regarded as the outcome of this study. It was
calculated as the interval between days from sHLH diagnosis and death
from any cause or the last follow-up. The survival status of all
participants was assayed through inpatient medical records and telephone
call.

\textbf{Treatment}

Specific treatment for sHLH was administrated to patients. Of 32 cases
in 25-(OH)-D \textless{}30nmol/L, 24 patients (75\%) received HLH-94 or
HLH-04 orsystemic combination chemotherapy like EPOCH, CHOP, DEP; 6
patients (18.8\%) received GC\selectlanguage{ngerman}± Ivig ± CsA alone; and 2 patients (6.3\%)
received only supportive therapy due to progressive multiple organ
dysfunction or patients' aspiration. Among 22 (40.7\%) patients with
25-(OH)-D\selectlanguage{english}[?]30nmol/L, 18 (81.8\%) were treated with chemotherapy or
HLH-94/04; 3 (13.6\%) were treated with GC+- Ivig +- CsA alone; and 1
patient (4.5\%) was administrated with supportive therapy. No
statistically significant difference was observed in treatment regimen
between two 25-(OH)-D groups (\emph{P} =0.839).

\textbf{Statistical Analysis}

All statistical analyses were performed using R software (version 3.6.3;
The R Foundation for Statistical Computing), SPSS (version 23, Chicago)
and STATA/MP(version 14, USA). Patients' continuous characteristics were
summarized as median+-quartile for non-normally distributed variables and
mean standard+- deviations (SDs) for normally distributed variables.
Categorical variables were presented as numbers and percentages.
Differences between VD groups were analyzed using wilcox and chi-square
tests, where appropriate. We conducted Kaplan-Meier method, univariable
Cox proportional hazards models and multivariable Cox model (variables
with \emph{P} \textless{}0.05) to estimate hazard ratios (HRs) and 95\%
confidence interval to discover independent prognostic variables on OS.
All subgroup analyses were presented with a COX test for interaction. A
two-sided \emph{P} \textless{} 0.05 was considered statistically
significant.

\textbf{Results}

\textbf{Baseline characteristics}

Table 1 displayed patients' clinical characteristics and laboratory
examination. The median age at diagnosis of total study population was
56 years and 23 patients (42.6\%) were defined old patients (over 60
years). Male occupied a large proportion (70.4\%), with ratio of 2.4.
More than half (53.7\%) suffered EBV infection. To be specific in terms
of clinical manifestations, 54 patients (100\%) presented with fever; 38
patients (70.4\%) manifested with splenomegaly. Other laboratory
findings of diagnostic criteria included hypofibrinogenemia (34/54),
hypertriglyceridemia (26/54), hyperferritinemia (52/54) and
hemophagocytosis (40/54).

The median level of 25-(OH)-D was 23.95 (range:7.5-90.8)ng/ml and 49
patients (90.7\%)had vitamin D deficiency (25-(OH)-D\textless{}50nmol/L,
i.e., 20ng/ mL). Then, we separated total cases into two groups
according to a published research: low 25-(OH)-D level (n=32,
\textless{}30 nmol/L) and relatively high 25-(OH)-D level (n=22, \selectlanguage{english}[?]30
nmol/L). Participants with lower 25-(OH)-D had higher TG (3.28\emph{vs.}
2.45mmol/L \emph{P} =0.032). There was no statistical difference on
other characteristics. Patients in the high 25-(OH)-D group had a higher
rate of hemophagocytosis, with a statistical trend (86.4\% \emph{vs.}
65.6\% \emph{P} =0.056).

According to the correlation analysis, the level of 25-(OH)-D was
opposite to triglycerides and ferritin, with significant importance
(Figure 1).

\textbf{Association with 25-(OH)-D and prognosis}

Upon final follow-up (February 1, 2021), the mortality rate was 83.3\%.
Median OS of the cohort group was 42 days (range: 1-669 days). As shown
in Figure 2, patients with low vitamin D level acquired lower overall
survival compared to those with high level (28 \emph{vs.} 125
days\emph{P} =0.041). In table 2, univariate KM or Cox analysis
demonstrated that hemoglobin, platelet, albumin, TG, \selectlanguage{greek}β\selectlanguage{english}2 -MG, ferritin,
log\textsubscript{e} (sCD25) was significantly predictive for survival
(\emph{P} \textless{}0.05). Then, we transferred those indexes
with\emph{P} \textless{} 0.05 into multivariate Cox regression analysis
to screen out independent risk factors of OS. The result demonstrated
that vitamin D was an independent factor for OS HR=0.364 ;95\% CI
0.183-0.724; \emph{P} = 0.004). Also, inferior survival engaged other
four factors:
log\textsubscript{e}(sCD25)(HR=2.291;95\%CI=1.186-4.426;\emph{P}
=0.014); \selectlanguage{ngerman}ß2-MG (HR=1.093;95\%CI=1.017-1.174;\emph{P} =0.015); platelet
(HR=0.983; 95\%CI=0.971-0.996; \emph{P} =0.008) .

\textbf{Significance of~25-(OH)D deficiency through~subgroup analysis}

Stratified subgroup analyses were conducted according to age (\selectlanguage{english}[?] 60
years, \textgreater{} 60 years), gender (male, female), EBV infection
status (negative, positive), pathogensis(MHLH,non-MHLH), hemoglobin
(\textless{}90g/L, \selectlanguage{english}[?]90g/L), platelet
(\textless{}100x10\textsuperscript{9}/L, \selectlanguage{english}[?]100x10\textsuperscript{9}/L),
Fib (\textless{}1.5 g/l, \selectlanguage{english}[?]1.5g/l), TG (\textless{}3mmol/L, \selectlanguage{english}[?]3mmol/L),
ferritin (\selectlanguage{english}[?]10000 ug/L, \textgreater{}10000 ug/L), sCD25 (\selectlanguage{english}[?]20000ng/l,
\textgreater{}20000ng/l). 25-(OH)-D had a significant interaction with
ferritin (\emph{P} =0.009), and vitamin D was an important protective
factor for ferritin\textgreater{}10000ug/L
(HR=0.052(0.005-0.590),\emph{P} =0.017). Although there were no
significant interactions for the associations between 25-(OH)-D and
survival across strata by other variables, vitamin D was more likely to
predictive of survival in older, male, MHLH, EBV infection,
hemoglobin\textless{}90g/L,
platelet\textless{}100x10\textsuperscript{9}/L, Fib \textless{}1.5g/L,
triglyceride, sCD25\textgreater{}20000ng/l group (Table 3).

\textbf{Discussion}

To the best of our knowledge, this was the first cohort study assessing
the prognostic role of vitamin D in secondary HLH. We discovered that 49
(90.7\%) sHLH suffered VD insufficiency (25-(OH)-D\textless{}50nmol/L).
Low 25-(OH)-D level was associated with survival of sHLH after adjusting
for other prognostic factors.

Vitamin D, one of the indispensable elements of health and disease
prevention, is mainly synthesized by the skin during sun exposure, or
ingested in daily diet. Measurement of serum 25(OH)D was capable of
represent the overall level of vitamin D in vitro \textsuperscript{20}.
According to previous research, vitamin D insufficiency is a widespread
healthy issue, more common in Asia\textsuperscript{21}. A large
multi-center study (2173 healthy adults in Dalian, Beijing, Hangzhou,
Guangzhou, and Urumqi) showed that 55.9\% populations had insufficient
vitamin D---serum 25(OH)D levels were less than 20
ng/ml\textsuperscript{22}. In immune-related diseases, the proportion of
VD insufficiency is higher or more serious. Schundeln et
al\textsuperscript{23} reported that the incidence in hemolytic anemia
was 80.5\%. 80\% of patients with multiple myeloma have
25-(OH)-D\textless{}50nmol/L\textsuperscript{24}. Consistent with those
studies about vitamin D in different diseases, VD insufficiency
incidence rate was over 90\% in HLH and 71.1\% patients were assigned to
vitamin D deficiency (\textless{}30nmol/L), suggesting vitamin D may be
the cause or rather a consequence of the disease. The result of a
meta-analysis showed circulating 25-(OH)-D contributed to the occurrence
of lung cancer, with increasing 10 nmol/L dose associated with 8\%
reduction in the risk\textsuperscript{25}.

The biological effects of vitamin D ranged from maintaining serum
calcium and skeletal homeostasis to modulating immune
processes\textsuperscript{26}. The latter may be one of the reasons
related to the prognosis of VD in sHLH. So far, the specific mechanisms
of HLH remains no consensus, but more researches considered that
congenital and acquired immunity contributed to the occurrence and
development. The excessive activation of
CD8\textsuperscript{+}lymphocytes and macrophages resulted in wild
secretion of pro- and anti-inflammatory cytokines, like IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-6,
IL-12, IL-16, IL-18, TNF-a, and IL-10; those in turn to trigger
persistent activation and cause the organ dysfunction
characteristics\textsuperscript{27}. Reichel et al \textsuperscript{28}
showed that the biologically active metabolite of vitamin D3
participated in modulating lymphocyte function via suppressing IL-2
secretion and decreasing production of IFN-\selectlanguage{greek}γ \selectlanguage{english}by normal human peripheral
blood lymphocytes. In addition, vitamin D is known to exert several
immunomodulatory effects on cytokines synthesis in vitro, as it inhibits
TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-12, and IL-17 and stimulates IL-10, promoting a shift from a
Th1 to a TH2 phenocyte\textsuperscript{29}. Furthermore, it increased
the expression of regulatory T lymphocytes with a skewing away from the
Th17 phenotype. It's noteworthy that 25-(OH)-D show a deeper
immunoregulatory effect after lymphocyte activation\textsuperscript{30}.
Therefore, 25-(OH)-D was considered to exert the beneficial effect on
protecting the risk of immuneregulated diseases. In our study, a
negative correlation between 25-(OH)-D and TG levels was observed
(r=-0.34, \emph{P} =0.011), while the increase in TG in sHLH was mostly
attributed to IFN-\selectlanguage{greek}γ. \selectlanguage{english}High levels of IFN-\selectlanguage{greek}γ \selectlanguage{english}increase very low-density
lipoprotein and decrease lipase activity in the posterior liver, leading
to TG clearance defects and hypertriglyceridemia\textsuperscript{31}.
25-(OH)-D was relatively associated with ferritin (\emph{P} =0.055).
Therefore,we speculated that VD may represent the level of IFN-\selectlanguage{greek}γ.
\selectlanguage{english}Overall survival was shorter in low 25-(OH)-D level group than that in
high level group (28 \emph{vs.} 125 days \emph{P} =0.041). After
adjusting for hemoglobin, platelet, albumin, TG, \selectlanguage{greek}β\selectlanguage{english}2-microglobulin,
ferritin, log\textsubscript{e}(sCD25), 25-(OH)-D was proved to be an
independent protective factor for overall survival (HR=0.364,
95\%CI=0.183-0.724, P=0.004). Fattizzo et al\textsuperscript{30}
reviewed that vitamin D deficiency is correlated with disease
severity/activity in autoimmune cytopenias. Another plausible pathway
reasonable for the relationship between 25-(OH)-D and HLH maybe the
mediation of iNK cells. Vitamin D receptor (VDR), the mediator of VD
activity, contributed to the development of invariant natural killer
(iNK) cells, a subset of lymphoid cells which participates in the host
immune response and limits autoimmunity in negative feedback
\textsuperscript{32}.

Second HLH is triggered by various pathologies, lymphoma, infections,
autoimmune disorders\textsuperscript{16}. Subgroup analysis showed that
in MHLH, vitamin D displayed remarkably ability in predicting survival
(HR=0.379, 95\%CI=0.187-0.769, \emph{P} =0.004). Lymphoma accounts for
98.1\% of malignant tumors in this study. In diffuse large B-cell
lymphoma (DLBCL) and T-cell lymphoma (TCL), Drake et
al\textsuperscript{33} found that the level of 25(OH)D and 1,25-(OH)-D
was directly predictive for OS (HR=1.99 95\% CI =1.27-3.13 for DLBCL;
HR=2.38 95\% CI=1.04-5.41 for TCL) and EFS (HR=1.41 95\%CI=0.98-2.04 for
DLBCL; HR=1.94 95\% CI=1.04-3.61 for TCL). Vitamin D can regulate
several key cellular processes, including inhibiting carcinogenesis by
inducing cell differentiation, inhibiting proliferation and
angiogenesis, and promoting apoptosis\textsuperscript{34}. VD also can
inhibit the proliferation of lymphoma cell lines and induce their
differentiation in vitro experiments\textsuperscript{35}. 25-(OH)-D was
proven to influence tumors microenvironment, where chemokines, chemokine
receptors, adhesion molecules interacted\textsuperscript{36}. Those may
explain the prognostic role of vitamin D in HLH.

Some limitations were present in our study. First, there was a
retrospective cohort study and the sample size was small. Then, further
clinical researches with more samples are needed to confirm our result.
Second, index including proinflammatory cytokines and/or inflammation
markers were absent to allow for the assessment of severity. Sfeir
et~al\textsuperscript{37} conducted a study about supplying vitamin D
and found the improvement of prognosis in lymphoma patients. However,
this study did not conclude the supplement of VD and answer whether
25-(OH) D supplement improve the therapeutic effect.

\textbf{Conclusion}

In general, vitamin D was significantly associated with survival outcome
and was adopted as a prognostic factor of HLH. We hope that clear oral
25-(OH)D supplement can receive more attention from clinicians.

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Figure 1: Correlation between 25-(OH)-D and neutrophil/lymphocyte ratio
(NLR), lymphocyte/monocyte ratio (LMR), hemoglobin (HB), platelet (PLT),
triglycerides (TG), albumin (ALB), calcium (Ca), \selectlanguage{ngerman}ß2- microglobulin
(ß2-MG), serum ferritin (SF), sCD25. R is the correlation coefficient.

Figure 2: Survival curves of different groups of 25-(OH)-D.

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