High concentration of miR-133 is a useful marker for the diagnosis of lymphoma- associated hemophagocytic syndrome

Abstract

BACKGROUND:

Lymphoma associated hemophagocytic syndrome (LAHS) is one of the major adult secondary hemophagocytic lymphohistiocytosis (HLH). Early diagnosis and treatment contribute to improved outcome. No enlarge lymph nodes can often delay the diagnosis of underlying lymphoma.

OBJECTIVE:

To find out criteria distinguishing LAHS from HLH induced by benign diseases.

METHODS:

clinical characteristic and laboratory feature of 31 patients with HLH (10 benign disease-associated HLH and 21 LAHS) were analyzed retrospectively.

RESULTS:

No significantly differences were observed in the levels of LDH, IL-6, IL-10, TNF- $\alpha$ ; however, the level of CRP (C reactive protein) and the mean level of sIL-R (soluble interleukin-2 receptor) were higher in patients with LAHS than those with benign disease associated disease associated HLH while ferritin levels were higher in benign disease associated HLH than in LAHS. Consequently, the serum sIL-2R/ferritin ratio of patients with LAHS was markedly higher than that of patients with benign disease associated HLH (0.33 $\pm$ 0.23 vs 5.82 $\pm$ 3.26, $P=$ 0.0001). In addition, we found out that the mean level of miR-133 (microRNA-133) was significant higher in LAHS than in benign disease associated HLH (18.83 $\pm$ 10.44 vs 5.82 $\pm$ 3.26, $P⩽$ 0.0001). Conclusion: Serum miR-133 is a new very useful marker for diagnosing of LAHS, but it need further confirmation by further clinical studies.

1. Introduction

Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder in adult characterized by persistent fever, splenomegaly with cytopenia (affecting 2 of 3 lineages in the peripheral blood), hypertriglyceridemia and/or hypo-fibrinogenemia, hemophagocytosis, hyperferritinemia, increased sIL-2R (soluble inter leukin-2 receptor), and low or absent natural killer(NK)-cell activity [1]. In the 1980s, the median survival rate of HLH was only 1–2 months, and the 1 year overall survival rate was on 5% [2]. Currently, HLH-94 (Hemophagocytic Lymphohistiocytosis Society-1994) or HLH-04 regimens are the standard HLH treatment strategies and have improved the prognosis, reaching a partial disease response in 76% and a complete disease response in 50–60% of cases [3, 4]. Lymphoma associated hemophagocytic syndrome (LAHS) is a subtype that account for approximately 40% of adult secondary HLH, and has a poor outcome compared to benign disease associated [5]. Early diagnosis of the underlying conditions, especially of lymphoma, may lead better outcome. That patients may not have enlarged lymph nodes renders the diagnosis of LAHS even more difficult. In this study, a new useful marker for early diagnosis of LAHS has been found out, and should confirmed by further investigations. MiR-133, encoded by more than one locus, is a member of myogenic miRNAs (myomiRs), which has two variants, miR-133a (miR-133a-1 and miR-133a-2) and miR-133b, were firstly identified in mice, but are located on different chromosomes depending on the species. MiR-133 has been found expression on lots of cancer, for example Hepatocellular carcinoma, NSCLC (non-small cell lung cancer), colorectal adenoma and cancer, ovarian cancer, glioblastoma, cervical carcinoma, and It has lots of function to inhibit or improve the cancer cell proliferation [6, 7, 8, 9, 10]. Some of research about how to diagnosis HLH trigger by lymphoma from other disease, one of the useful marker is high sIL-R/Ferritin ratio. Whether other factor can do as this marker, so we do this research.

2. Methods

2.1 Patients and methods

We retrospectively collected data of 31 patients who were diagnosed HLH (LAHS and benign disease associated HLH) from 2011 to 2016 at Jiangxi Cancer Hospital, The Second Affiliated Hospital to Nanchang University and The First Affiliated Hospital to Nanchang University. Data collected on each patient included age, presumed etiology, Splenomegaly, white blood cell (WBC), absolute neutrophil count (ANC), hemoglobin (Hb), platelet (PLT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), triglycerides (TG), fibrinogen (Fib), C-reactive protein (CPR), ferritin, bone marrow nuclear cell count (NCC), tumor necrosis factor $\alpha$ (TNF- $\alpha$ ), Total bilirubin (TBIL), Indirect bilirubin (IBIL)miR-133 and percentage of macrophages in the bone marrow. HLH was diagnosed according to the HLH 2004 diagnostic guidelines. Five of the following eight criteria had to be fulfilled. (1) Fever; (2) Splenomegaly; (3) Cytopenia (affecting $⩾$ 2 of 3 lineages in the peripheral blood): Hemoglobin $<$ 90 g/L (in infants $<$ 4 weeks: hemoglobin $<$ 100 g/L)Platelets $<$ 100 $\times$ 10 ${}^9$ /L Neutrophils $<$ 1.0 $\times$ 10 ${}^9$ /L (4) Hypertriglyceridemia and/or hypofibrinogenemia: fasting triglycerides $⩾$ 3.0 mmol/L ( $⩾$ 265 mg/dl) Fibrinogen $⩽$ 1.5 g/L; (5) Hemophagocytosis in bone marrow or spleen or lymph node No evidence of malignancy (6) Low or absent NK-cell activity (according to local laboratory reference; (7) Ferritin $⩾$ 500 $\mu$ g/L; (8) Soluble CD25 (soluble IL-2 receptor) $⩾$ 2,400 U/ml. Quantitative real-time PCR was performed on 7500 Fast Real-Time PCR System. The SYBR Green PCR Master Mix Kit (Applied Bio-systems, Foster City, CA) was used for relative quantification of miRNAs with U6 as an internal control. The primers used for quantitative real-time PCR were follow: miR-133: forward 5 ${}^{\prime }$ forward 5 ${}^{\prime }$ -GGGTTTGGTCCCCTTCAA-3 ${}^{\prime }$ , reverse 5 ${}^{\prime }$ -AGTGCGTGTCGTGGAGTC-3 ${}^{\prime }$ . The PCR cycles consisted of denaturation at 95 ${}^{\circ }$ C for 15 seconds, and annealing as well as extension at 60 ${}^{\circ }$ C for 1 minute for 40 cycles. Each sample was analyzed two times. The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence exceeds the given threshold. The obtained data were translated into log2 scale. The 2- $\mathrm{\Delta }\mathrm{\Delta }$ Ct method was used to analyze the relative expression of miRNAs. The diagnosis of malignant lymphoma was mad according to the 2008 WHO classification. We compared the laboratory findings of patients with LAHS to laboratory findings of patients with benign disease associated HLH. The nonparametric Mann-Whitney U test was used to compare the values of the two groups. In all test, a $P$ value of $<$ 0.05 was considered significant.

3. Results

The median age of the benign disease-associated HLH group (Tables 1 and 2) was 48.9 years, ranging from 18 to 77 years. The etiologic subtypes were infection (Epstein-Barr virus, $n=$ 2; Tuberculosis, $n=$ 2, Cytomegalovirus, $n=$ 1), autoimmune disorders. (SLE, $n=$ 2, adult onset Still’s disease, $n=$ 3). After immunosuppressive therapy and treating the underlying disorder, 8 of the ten patients recovered, two patients died with multiple system organ failure.

Table 1

Clinical characteristic of patients with benign disease associated HLH

				WBC	ANC	Hb	PLT	AST	ALT	LDH	TG	Fib	CRP
Case	Age	Sex	Cause	(/ $\mu$ l)	(/ $\mu$ l)	(g/dl)	(/ $\mu$ l)	(U/l)	(U/l)	(/UNL)	(mg/dl)	(g/l)	(mg/l)
P1	18	M	AOSD	2400	380	8.0	3.1	700	650	2.2	200	1.2	1.5
P2	25	F	SLE	3000	1100	10	4.2	300	347	3	199	1.5	30
P3	57	m	TB	1000	300	9.7	4.3	180	451	2.7	15	0.9	10
P4	36	M	TB	2000	600	8.6	5.7	330	379	3.1	729	1.3	8.9
P5	45	M	AOSD	1500	450	11	4.9	432	531	2.2	541	2.3	10.9
P6	49	F	SLE	2400	500	12.3	2	522	620	3.0	207	1.4	24.3
P7	77	F	CMV	800	200	11.5	1	345	337	3.7	178	0.3	10.7
P8	54	M	EBV	750	150	8.8	8	226	259	1.5	521	0.7	22.1
P9	67	M	EBV	400	100	9.2	3.3	270	325	2.9	455	0.66	15.4
P10	61	M	AOSD	3200	1120	10.7	5.2	170	220	1.7	412	1.2	13.3

ANC absolute neutrophil count, TG triglyceride, Fib fibrinogen, CMV cytomegalovirus, EBV Epstein-Barr virus, AOSD adult onset Still’s disease, UNL upper normal limit.

Table 2

Clinical characteristics of patient benign disease associated HLH

Case	Splenomegaly	Hemophagocytosis	NCC	Histiocyte	HLH-2004	sIL-2R	Ferritin	sIL-2R/Ferritin	miR-133
		(bone marrow)	(/ $\mu$ l)	(%)		(U/ml)	(ng/ml)		(copy/ml $\times$ 10 ${}^3$ )
P1	$+$	$+$	44.1 $\times$ 10 ${}^4$	0.5	5(8)	2251	27300	0.08	1.3
P2	$+$	$+$	3.1 $\times$ 10 ${}^4$	1.5	7(8)	2345	12451	0.19	0.9
P3	$+$	$+$	7.4 $\times$ 10 ${}^4$	6.0	6(8)	1976	5871	0.34	2.1
P4	$+$	$+$	6.6 $\times$ 10 ${}^4$	2.5	5(8)	3359	10362	0.32	3.0
P5	$+$	$-$	2.3 $\times$ 10 ${}^4$	3.1	7(8)	8766	17302	0.51	1.7
P6	$+$	$+$	5.5 $\times$ 10 ${}^4$	1.0	5(8)	7022	8102	0.87	0.5
P7	$+$	$+$	0.8 $\times$ 10 ${}^4$	2.0	5(8)	7515	23057	0.33	6.0
P8	$+$	$+$	1.3 $\times$ 10 ${}^4$	4.0	5(8)	2451	10458	0.23	1.93
P9	$+$	$+$	2.4 $\times$ 10 ${}^4$	4.1	5(8)	4513	15279	0.30	2.3
P10	$+$	$+$	1.7 $\times$ 10 ${}^4$	3.8	7(8)	2201	16597	0.13	2.46

NCC: bone marrow nuclear cell count.

3.1 Clinical features

Total of 31 patients were included in this research. The clinical characteristics of these patients are described in Tables 1–4. Ten benign diseases associated HLH is in Tables 1 and 2; Twenty-one LAHS patients is in Tables 3 and 4.

The median age of the patients with HLH was 50.8 years (Tables 3 and 4), ranging between 19 to 77 years. The etiologic subtype is 3 HD and NHL 18 (PTCL 7, DLBCL 5, MCL 1, NK/T 4, FL 1). Three patients is the first time diagnosis lymphoma, and others are relapse or refractor and all patients were clinical stage IV and have B symptom, and bone marrow involvement was observed in 10 patients. According to the International Prognostic Index, all patients were categorized as media to high risk group.

Seven of these patients with LASH have no distinct enlarge lymph nodes. Lymphoma was diagnosed by biopsy from liver 2, testis 1, spleen 1, nasal pharynx 2 or skin of the lesion 1.

The laboratory findings of the initial presentation are summarized in Tables 1 and 2 (case p1–10, the group patients with benign disease associated HLH) and Tables 3 and 4 (case L1–21, the LAHS group). All our patients fulfilled the required criteria for the diagnosis of HLH. All patients have high serum ferritin levels, and only 2 patients had not found out hemophagocytosis in bone marrow, the other ten patients have hemophagocytosis. Nineteen patients had anemia (Hb $<$ 10.0/dl), but all patients had thrombocytopenia (PLT $<$ 100,000/ $\mu$ l). We also find out that increased levels of LDH, AST, ALT, ferritin, CRP, sIL-2R and miR-133 in all patients examined. 11 patients had hypofibrinogenemia (Fibrinogen $<$ 150 mg/dl), and 21 patients had hypertriglyceridemia (triglycerides $>$ 265 mg/dl).

3.2 Comparison of laboratory findings between LAHS and benign disease associated HLH

A comparison of laboratory findings between the LAHS and benign disease associated HLH group in shown in Table 5. There were no significant differences in LDH or ALT and AST levels ( $P=$ 0.82, 0.06 and 0.242). But the level of CRP is higher in LAHS than that of in benign associated disease associated HLH ( $P=$ 0.001), most of LAHS with fever unknown origin so the CRP is higher than benign disease associated HLH. WBC and Neutropenia and thrombocytopenia were more severe in the benign disease associated HLH group, but no significant differences ( $P=$ 0.272). Anemia was more severe in the LAHS group, it also no significant differences too ( $P=$ 0.382).

Table 3

Clinical characteristics of patients with LAHS

Case	A	S	Cau	DS	BM	ST	WBC/	ANC/	Hb	PLT/	AST	ALT	LDH/	Fib	TG	CRP
							$\mu$ L)	$\mu$ L	g/dl	$\mu$ L	U/l	U/L	UNL	g/l	mg/dl	mg/dl
L1	25	F	MCL	LN	N	IV	1245	280	8.6	5.2	102	115	4	1.0	277	26
L2	19	M	HD	LN	N	IV	1350	300	1.2	1.1	117	123	0.7	0.5	256	17
L3	47	M	DLBCL	LV	Y	IV	1500	100	5.8	1.5	132	120	1.6	0.8	520	24
L4	54	F	NK/T	NA	N	IV	1590	400	7.6	1.7	157	231	1.6	1	457	31
L5	39	F	DLBCL	LN	Y	IV	1600	500	7.7	2	170	150	1.9	0.9	359	29
L6	71	M	PTCL	LV	Y	IV	1700	1100	7.9	2.1	178	145	1.9	0.9	456	32
L7	63	F	PTCL	LN	Y	IV	1800	300	7.9	2.2	198	206	4.9	1.2	736	43
L8	45	F	NK/T	NA	N	IV	1900	300	8.3	3.3	200	213	2.1	1.8	n.t.	33
L9	58	F	PTCL	LN	N	IV	2000	400	8.8	3.8	220	219	1.9	1.2	212	47
L10	64	M	DLBCL	LN	Y	IV	2100	700	8.7	3.9	231	154	3.7	1.3	453	57
L11	73	M	DLBCL	SP	N	IV	2150	700	9.2	4	712	234	2.1	1.4	219	52
L12	77	F	FL	LN	Y	IV	2300	10000	92	5.5	541	271	3.0	1.45	324	51
L13	19	F	HD	LN	N	IV	2400	700	9.4	5.7	426	234	1.9	1.8	298	57
L14	26	M	PTCL	LN	Y	IV	2900	6000	9.7	6.2	330	289	4.9	1.5	513	62
L15	73	M	DLBCL	TE	Y	IV	3000	900	10.2	6.3	378	330	3.7	1.9	221	57
L16	33	F	HD	LN	Y	IV	3000	600	10.3	7.2	319	231	2.5	2.2	312	61
L17	39	F	PTCL	LN	Y	IV	3000	400	11.7	8.3	237	376	2.7	2.1	375	73
L18	51	M	NK/T	LN	N	IV	3400	1340	12.4	7.5	300	425	2.9	2.2	297	92
L19	44	F	PTCL	LN	Y	IV	3500	1100	12.5	7.6	318	451	2.4	2.8	n.t.	63
L20	71	M	NK/T	SK	N	IV	4200	600	12.7	8.3	312	291	2.5	3	764	24
L21	69	M	PTCL	LN	N	IV	8000	1100	13	9.7	246	600	2.2	3.2	425	135

A age, S sex, DS diagnosis site, ANC absolute neutrophil count, Cau cause, ST stage, BM bone marrow invasion, TG triglyceride, Fib fibrinogen, DLBCL NOS diffuse large B cell lymphoma, UNL upper normal limit, PTCL NOS peripheral T cell lymphoma, NK/T nature killer/T cell lymphoma, MCL mantle cell lymphoma, HD Hodgkin disease, FL follicular lymphoma, LN lymph node, SK skin, LV liver, SP spleen, NA nasal.

Table 4

Clinical characteristics of patients with LAHS

Case	Splenomegaly	Hemophagocytosis	NCC	Histiocyte	HLH-2004	sIL-2R	Ferritin	sIL-2R/Ferritin	miR-133
		(bone marrow)	(/ $\mu$ l)	(%)		(U/ml)	(ng/ml)		(copy/ml $\times$ 10 ${}^3$ )
L1	$+$	$+$	12.1 $\times$ 10 ${}^4$	0.7	5(8)	9517	1347	7.07	15
L2	$+$	$+$	2.1 $\times$ 10 ${}^4$	1.8	7(8)	5546	1800	3.08	290
L3	$+$	$+$	5.4 $\times$ 10 ${}^4$	6.3	6(8)	6671	3056	2.18	11
L4	$+$	$+$	3.6 $\times$ 10 ${}^4$	2.7	5(8)	15432	1162	13.28	13
L5	$+$	$+$	4.3 $\times$ 10 ${}^4$	2.1	7(8)	8987	2104	4.27	270
L6	$+$	$+$	1.5 $\times$ 10 ${}^4$	4.0	5(8)	7213	1357	5.32	5.5
L7	$+$	$+$	1.8 $\times$ 10 ${}^4$	1.0	5(8)	8786	3057	2.87	23.2 ${}^4$
L8	$+$	$+$	2.3 $\times$ 10 ${}^4$	4.9	5(8)	12541	1423	8.81	110
L9	$+$	$+$	0.94 $\times$ 10 ${}^4$	4.3	5(8)	25421	3201	7.94	13
L10	$+$	$+$	1.7 $\times$ 10 ${}^4$	2.8	5(8)	7952	1597	4.98	14.6
L11	$+$	$+$	1.8 $\times$ 10 ${}^4$	3.2	6(8)	3421	1754	1.95	32.1
L12	$+$	$-$	2.3 $\times$ 10 ${}^4$	1.07	6(8)	6541	2543	2.57	26.9
L13	$+$	$+$	1.7 $\times$ 10 ${}^4$	6.7	5(8)	5761	1200	4.8	5.7 $\times$
L14	$+$	$+$	0.7 $\times$ 10 ${}^4$	5.1	6(8)	5642	1798	3.13	9.78
L15	$+$	$+$	3.2 $\times$ 10 ${}^4$	0.2	5(8)	10531	807	13.04	31.4
L16	$+$	$+$	2.7 $\times$ 10 ${}^4$	0.7	5(8)	7621	1345	5.63	26.7
L17	$+$	$+$	4.5 $\times$ 10 ${}^4$	3.1	5(8)	7239	907	7.98	34.9
L18	$+$	$+$	9.2 $\times$ 10 ${}^4$	1.4	5(8)	12045	1338	9.00	9.43
L19	$+$	$+$	20.1 $\times$ 10 ${}^4$	1.5	6(8)	9542	3120	3.05	30
L20	$+$	$+$	11.7 $\times$ 10 ${}^4$	0.9	7(8)	9733	1703	5.72	41.3
L21	$+$	$+$	30.2 $\times$ 10 ${}^4$	1.1	6(8)	12692	2307	5.5	72.8

NCC: bone marrow nuclear cell count.

Table 5

Comparison of the laboratory findings of the two groups (benign disease associated HLH and LAHS)

	Benign disease associated HLL	Range		LAHS	Range		$P$
	Mean $\pm$ SD			Mean $\pm$ SD
	$N=$ 10			$N=$ 21
WBC ( $\times$ 10 ${}^9$ )	1745.00 $\pm$ 995.67	400	–3200	2601.67 $\pm$ 1470.56	1245	–8000	0.107
ANC ( $\times$ 10 ${}^9$ )	490.00 $\pm$ 362.52	100	–1120	639.05 $\pm$ 338.97	100	–1340	0.272
Hb (g/dl)	9.98 $\pm$ 1.38	8	–12.3	9.18 $\pm$ 2.66	1.20	–13.00	0.382
PLT ( $\times$ 10 ${}^9$ )	4.17 $\pm$ 1.98	1.00	–8.00	5.05 $\pm$ 2.87	1.10	–11.30	0.389
AST (U/L)	347.50 $\pm$ 164.91	170	–700	277.33 $\pm$ 147.02	102	–712	0.242
ALT (U/L)	411.90 $\pm$ 146.96	220	–650	262.76 $\pm$ 124.69	115	–600	0.006
LDH (/UNL)	2.60 $\pm$ 0.68	1.50	–3.70	2.52 $\pm$ 0.95	0.7	–4.9	0.812
CRP (MG/DL)	14.71 $\pm$ 8.46	1.5	–30	52.81 $\pm$ 26.43	17	–135	0.00*
sIL-2R (U/ml)	4039.10 $\pm$ 2820.60	225	–8766	9468.29 $\pm$ 4641.83	3421	–25421	0.002*
Ferritin (ng/ml)	14677.90 $\pm$ 6688.75	5871	–27300	1853.19 $\pm$ 750.77	807	–3201	0.000*
sIL-2R/Ferritin	0.33 $\pm$ 0.23	0.08	–0.87	5.82 $\pm$ 3.26	1.95	–13.28	0.000*
miR-133 (copy $\times$ 10 ${}^3$ )	2.22 $\pm$ 1.52	0.5	–6.0	18.83 $\pm$ 10.44	4.13	–34.9	0.001*

UNL upper normal limit, NS not significant; *significant.

The mean sIL-2R level was higher (4039.10 $\pm$ 2820.60 vs 9468.29 $\pm$ 4641.83, $P=$ 0.002), and the mean ferritin levels was lower (1853.19 $\pm$ 750.77 vs 14677.90 $\pm$ 6688.75, $P=$ 0.001) in the LAHS group than those in the benign disease associated HLH. We also found out that the copy of miR-133 in LAHS is higher than benign disease associated HLH ( $P=$ 0.0001), respectively (Table 5).

4. Discussion

Secondary HLH presents in number of different clinical conditioning with various etiologic associations [11]. Patients may have concurrent infections or other conditions that trigger their HLH, such as malignancy or autoimmune diseases [12]. We retrospectively 31 secondary HLH cases at three medical center of China.

The major conditions of these patients are lymphoma, followed by infection (EBV, MCV, TB) and autoimmune disorders. These 10 benign disease associated HLH show 50% have autoimmune disorder, and others were infection disease, some researches show the same ratio of patients with autoimmune disorder too [13], the secondary HLH in childhood is different from adult that most trigger is infection and the virus infection is the first conditions for triggering HLH [14]. But adult onset secondary HLH most have malignancy disease, for example lymphoma and leukemia is the most condition and other malignancy [15, 16]. Hematology malignancy is the most conditions for adult secondary HLH, and most of them have poor outcome, especially the outcome of patients with LAHS is the poorest. Seventy percent patients with LAHS is at the end-stage of disease, these patients are multiple-drugs resistance and will died in several months. The out of patients who present with LAHS as the first symptom is poorer than the secondary LAHS [17]. In the LAHS group, approximate 52% (10/21) patients were associated with B cell lymphoma (HD 3, DLBCL 6, FL 1, MCL 1), so our result shows that B cell lymphoma is the most trigger for LAHS, it is similarity with other research. PTCL is the second lymphoma trigger for LAHS 33% (7/21), and almost all patients who have second LAHS were fever of unknown origin, 2/7 have skin damage, first diagnosis dermatology, final skin biopsy diagnosis PTCL and treatment with chemotherapy, then the fever was control.

In two recent investigations, a sIL-2R/ferritin ration $>$ 2 has been observed in most patients with LAHS and only rarely in those with benign disease associated HLH. Therefore, a high sIL-2R/ferritin ratio has been proposed as a useful marker for the diagnosis of a LASH [18, 19]. These results were confirmed by our study as 20 out of 21 patients with LAHS had a sIL-2R/ferritin ration $>$ 2 and all 10 patients with benign disease associated HLH had a sIL-2R/ferritin ration $<$ 2 (Table 5). High levels of ferritin are one of the changing of iron overload; in some conditions iron overland impact the outcome of diseases. Umit [20] research show ferritin $>$ 2000 plus TS (TS was calculated by dividing serum iron level to total iron binding capacity) $>$ 45% was found to have an association with VOD (veno-oclussive disease) at borderline significance in patients post hematopoietic stem cell transplantation. In our research, the result show the patients who with benign disease associate HLH have higher level of ferritin than those with LAHS. CD47 down regulation that increases hematopoietic stem cell engulfment by macrophages causing cytopenia was reported to be critical in the pathogenesis of (HLH) [21]. MiR-133 can down regulation the expression of CD47 on hematopoietic stem cell. We have check the level of miR-133 in the serum of patients who with LAHS and in those with benign disease triggering HLH and have found out that the level of miR-133 are higher in LAHS than in benign disease associated HLH (Table 5). We also detected the concentration of miR-133 in patients with lymphoma without HLH, and the values were similar with those of benign disease associated HLH. A high concentration of miR-133 may be one of factors to distinguish LAHS from benign disease triggering HLH and this finding should be confirmed by further studies.

Conflict of interest

The authors declare no competing financial interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.