Notch 1 and NF-κB Expression and Clinical Correlation in Chinese Patients with Lymphoblastic Lymphoma

doi:10.4236/jct.2013.43A053

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Journal of Cancer Therapy, 2013, 4, 441-447

http://dx.doi.org/10.4236/jct.2013.43A053 Published Online March 2013 (http://www.scirp.org/journal/jct)

441

Notch 1 and NF-κB Expression and Clinical Correlation in

Chinese Patients with Lymphoblastic Lymphoma

Lin Lin1,2, Xiaofei Sun1,2*, Juan Wang1,2, Zijun Zhen1,2, Suxia Lin1,3*, Gangling Tong1,2,

Yan Chen1,2

1State Key Laboratory of Oncology in South China, Guangzhou, China; 2Department of Pediatric Oncology, Sun Yat-Sen University

Cancer Center, Guangzhou, China; 3Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China.

Email: *[email protected], *[email protected]

Received December 23rd, 2012; revised January 25th, 2013; accepted February 4th, 2013

ABSTRACT

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LBL) is commonly associated with Notch 1 mutations.

There is limited data on the relationship between Notch l and NF-κB expression and clinical features in LBL. We

evaluated the expression of Notch l and NF-κB in LBL using immunohistochemistry and analyzed their relationship

with clinical characteristics, treatment results, and survival. From October 2000 to August 2008, 34 untreated patients

with LBL were enrolled in the study. Median age was 11.8 years (range, 1 - 25 years). Twenty-five patients were diag-

nosed with T-LBL and 9 patients with B-LBL. Most patients received chemotherapy consisting of modified ALL-BFM-

90. Notch l showed high expression in 68% of T-LBL and low expression in 100% of B-LBL (p = 0.015). High expres-

sion of Notch l positively correlated with presence of a mediastinal mass but not with 5-year event free survival (EFS)

in T-LBL. NF-κB showed high expression in 65% of all patients with LBL, with no difference between T- and B-LBL.

NF-κB expression was higher in T-LBL patients with bulky disease and B symptom; it did not correlate with 5-year

EFS in T-LBL. Expression of Notch 1 and NF-κB strongly correlated (p = 0.014) in T-LBL. Notch 1 is highly ex-

pressed in T-LBL. NF-κB is highly expressed in all patients with LBL with no difference between T-LBL and B-LBL.

Notch 1 expression was significantly associated with NF-κB expression in T-LBL. Notch l and NF-κB may play an im-

portant role in the development of T-LBL; further investigation is warranted.

Keywords: Lymphoblastic Lymphoma; Notch l; NF-κB

1. Introduction

Lymphoblastic lymphoma (LBL) is a highly invasive

malignancy originating from immature precursor lym-

phocytes. It is divided into precursor T cell lymphoblas-

tic lymphoma (T-LBL) and precursor B cell lymphoblas-

tic lymphoma (B-LBL). LBL and acute lymphoblastic

leukemia (ALL) share common morphologic and im-

munophenotypic features as well as a favorable outcome

after ALL-type chemotherapy; they are thought to repre-

sent a spectrum of a single disease entity, which is

termed T (or B) lymphoblastic leukemia/lymphoma in

the World Health Organization (WHO) [1] classification

schemes. Children and adolescents with LBL have better

survival when treated with intensive chemotherapeutic

regimens used in ALL, with a 5-year event free survival

of 70% - 90% [2,3]. However, 5-year event free survival

is less than 50% in high risk and refractory patients in

spite of intensive chemotherapy. New biological markers

and targeted therapies are need for these refractory LBL

patients.

In 1991, human Notch 1 was identified at the chromo-

somal breakpoint of a subset of T lymphoblastic neo-

plasm at the t (7;9) (q34;q34.3) chromosomal transloca-

tion [4]. Mammals have four Notch receptors (Notch 1 -

4) [5]. Notch l is a member of the Notch transmembrane

bound receptor family. Being a signaling pathway, Notch

1 not only plays an important part in differentiation and

development of normal cells, but also in the germination

and growth of neoplastic lesions, and as a suppressor

and/or promoter in various tumors [5,6]. Many studies

report Notch 1 mutations in greater than 50% of human

T-ALL cases [7-10]. G-secretase inhibitors may inhibit

the growth of T-ALL through blockage of the Notch 1

signal pathway [11]. Activation of Notch 1 leads to the

development of T-ALL in a mouse model [12].

NF-kappa B (NF-κB) was identified in B lymphocytes

in 1986. It acts as a regulator of κB light chain expres-

sion in B cells, and was thus termed nuclear transcription

factor-κB (NF-κB) [13]. NF-κB can promote the germi-

nation, proliferation, invasion, and metastasis of tumors

*Corresponding author.

Notch 1 and NF-κB Expression and Clinical Correlation in Chinese Patients with Lymphoblastic Lymphoma

442

through regulation of gene transfer [14]. The NF-κB path-

way is a major downstream target of Notch 1 in T-cell

leukemia and is highly active in established human T-

ALL. Inhibition of this pathway can efficiently restrict

tumor growth both in vitro and in vivo. Thus NF-κB is

one of the major mediators of Notch 1-induced transfor-

mation, suggesting that the NF-κB pathway is a potential

target for future T-ALL therapies [15].

The above mentioned studies focused on T-ALL and

less on LBL. Although ALL and LBL are the same dis-

ease entity, they show some differences in clinical char-

acteristics. The expression of Notch-1 and NF-kappa B in

precursor lymphoid neoplasms has been widely explored.

However, the data regarded mainly leukemic presenta-

tion while studies on lymphoblastic lymphoma are scarce.

Our research aimed to detect expression of Notch l and

NF-κB in tissues of LBL and normal lymph nodes using

immunohistochemistry. We also analyzed the relation-

ship between expression of these markers and clinical

characteristics and prognosis, aiming to provide evidence

for their potential use as biological markers.

2. Materials and Methods

2.1. Materials

From October 2000 to August 2008, the tumor samples

from 34 untreated patients with lymphoblastic lymphoma

at Sun Yat-Sen University Cancer Center were obtained.

Patients included 30 males and 4 females with a median

age of 11.8 years (range, 1 - 25 years; 1 - 18 years: 29

cases; 18 - 25 years: 5 cases). Twenty-five patients were

diagnosed with T-LBL and 9 with B-LBL. According to

the St. Jude staging system, our patient sample include 1

stage I, 4 stage II, 12 stage III, and 17 stage IV patients.

Nine patients had B symptoms, 9 had large masses, 17

had bone marrow involvement, 22 had an anterior medi-

astinal mass, 9 had extranodal lesions, and 20 had in-

creased serum lactate dehydrogenase (LDH) levels.

Thirty-one patients were treated according to the modi-

fied ALL-BFM-90 protocol, 2 patients received CHOAP,

and 1 patient abandoned treatment. Five normal lymph

nodes were obtained from October 2007 to October 2008

to serve as a control group. The informed consent of pa-

tients or their guardian had been obtained.

2.2. Immunohistochemistry

The tissue was stained by a two-step immunohistochem-

istry staining method. The first antibody for Notch 1

staining was goat anti-human antibody from Santa Cruz

(working concentration 1:100) and the first antibody for

NF-κB staining was mouse anti-human antibody from

Santa Cruz (working concentration 1:150). The second

antibody for both stains was derived from an instant de-

tection kit (PV-6003) from Zhongshan Goldenbridge Bio-

technology Co., Ltd.

2.3. Microscopic Evaluation

Using the semi-quantitative evaluation method by Car-

cangiu et al. [16], membrane, plasma, and nuclear details

of tumor cell were observed microscopically and evalu-

ated for staining degree and number of positive cells. The

staining degree of cells was defined as colorless, light tan,

tan, or brown and was assigned a score of 0, 1, 2, or 3,

respectively. The number of positive cells was defined as

<5%, 5% - 35%, 36% - 70%, or >70% and was assigned

a score of 0, 1, 2, or 3, respectively. Results were as-

sessed by the product of the two scores as follows: ≤1 as

negative (−), 2 - 3 as weak positive (+), 4 - 5 as median

positive (++), and ≥6 as strong positive (+++). Negative

and weak positive (+) were considered low expression,

and median positive (++) and strong positive (+++) were

considered high expression.

2.4. Follow-Up

Follow-up studies were performed at three-month inter-

vals after ending maintenance therapy during the first

year, six-month intervals during the second year, and

one-year intervals during the following three years.

Event free survival (EFS) was calculated from the first

day of chemotherapy to death due to any cause, relapse,

progression, second malignancy, or the date of the last

follow-up contact for patients who were alive.

2.5. Statistical Analysis

Differences between two sample rates were analyzed

with the Fisher exact test two-sided for discrete variables.

The correlation between two nonparametric samples was

analyzed with spearman correlation test. Kaplan-Meier

test was implemented in survival analysis. The statistical

analysis was performed using SPSS statistics (version

16.0; SPSS). P values were considered significant at less

than 0.05.

3. Results

3.1. Expression of Notch-1 and NF-Kappa B in

T-LBL Compared to B-LBL

Notch 1 protein was expressed in the cytomembrane and

cytoplasm. High expression of Notch-1 was found in

50% (17/34) of all LBL cases. In T-LBL, high expression

was seen in 68% (17/25) and low expression in 32%

(8/25). In B-LBL, 100% (9/9) showed low expression.

Five normal lymph nodes also showed low expression.

Expression of Notch l was significantly different between

T-LBL and B-LBL (p = 0.001); expression was also sig-

Notch 1 and NF-κB Expression and Clinical Correlation in Chinese Patients with Lymphoblastic Lymphoma

443

nificantly different when comparing T-LBL and normal

lymph nodes (p = 0.009). B-LBL and normal lymph

nodes all showed low expression of Notch 1 (Table 1

and Figure 1).

NF-κB protein was expressed in the cytoplasm and

nucleus. Low expression was seen in 35% (12/34) and

high expression in 65% (22/34) of all LBL cases. In

T-LBL, low expression occurred in 32% (8/25) and high

expression in 68% (17/25). In B-LBL, low expression

was seen in 44% (4/9) and high expression in 56% (5/9).

There was no difference in NF-κB expression between

T-LBL and B-LBL, p = 0.687 (Table 2 and Figure 2).

We analyzed correlation of Notch-1 and NF-κB ex-

pression in T-LBL and found 14 cases with high expres-

sion of Notch1 also presented with high expression of

NF-κB. Notch 1 expression correlated significantly with

NF-κB expression in T-LBL, p = 0.014, no correlated

was seen in B-LBL (Table 3).

3.2. Association of Notch-1 and NF-κB

Expression with Clinical Characteristics of

T-LBL

We analyzed T-LBL by one factor correlation analysis

and found that high Notch l expression correlated sig-

nificantly with the presence of a mediastinal mass (p =

0.046). However, Notch l expression did not significantly

correlate with gender, age, extranodal lesions, bulky dis-

ease, LDH level, or presence of B symptoms (p > 0.05).

NF-κB expression was positively related to bulky disease

and B symptom (p = 0.013 and 0.000, respectively).

NF-κB expression did not correlate with other clinical

Table 1. Expression of Notch 1 in LBL and normal lymph nodes.

Low expression High expression

Samples Case (n)

Case (n) Percent Case (n) Percent p-value

T-LBL 25 8 32 17 68

B-LBL 9 9 100 0 0

0.001

T-LBL 25 8 32 17 68

Normal lymph nodes 5 5 100 0 0 0.009

B-LBL 9 9 100 0 0

Normal lymph nodes 5 5 100 0 0 No

Table 2. Expression of NF-κB in LBL and normal lymph nodes.

Low expression High expression

Case (n)

Case (n) Percent Case (n) Percent p-value

T-LBL 25 8 32 17 68

B-LBL 9 4 44 5 56

0.687

T-LBL 25 8 32 17 68

Normal lymph nodes 5 5 100 0 0 0.009

B-LBL 9 4 44 5 56

Normal lymph nodes 5 5 100 0 0 0.086

Table 3. Correlation of Notch-1 and NF-κB expression in T-LBL and B-LBL.

NF-κB expression

Notch 1 expression − + ++ +++

R value p value

T-LBL − 0 2 2 0

+ 1 2 1 0

++ 3 0 1 3

+++ 0 0 6 4

0.485 0.014

B-LBL − 2 0 3 0

+ 1 1 0 2

++ 0 0 0 0

+++ 0 0 0 0

6.3

0.098

Notch 1 and NF-κB Expression and Clinical Correlation in Chinese Patients with Lymphoblastic Lymphoma

444

Figure 1. Notch 1 expression in lymphoblastic lymphoma

(400×). (A) Low expression of Notch1; (B) High expression

of Notch1.

Figure 2. NF-κB expression in lymphoblastic lymphoma

(400×). (A) Low expression of NF-κB; (B) High expression

of NF-κB.

characteristics (Table 4).

3.3. Relationship between Notch 1 and NF-κB

Expression and Survival in T-LBL

In the 25 patients with T-LBL, median follow up was 55

months (range, 11 - 142 months). Twenty-one patients

were alive and 4 had died from disease. Five-year event

free survival was 82% ± 8.1%. In the Notch l high ex-

pression and low expression groups, 5-year event free

survival rates were 80.2% ± 10% and 87.5% ± 11%, re-

spectively (p = 0.80) (Figure 3). The 5-year event free

survival in the NF-κB high expression and low expres-

sion groups were 86.3% ± 9% and 75.0% ± 15%, respec-

tively (p = 0.32) (Figure 4).

4. Discussion

Deregulated Notch signaling has been implicated in

many diseases, but the clearest example of a pathogenic

role is found in T-cell lymphoblastic leukemia/lym-

phoma, where the majority of human and murine tumors

have acquired mutations that lead to aberrant increase in

Notch 1 signaling. Many studies report Notch 1 muta-

tions in greater than 50% of human T-ALL/T-LBL cases

[7-10]. Most of these studies detected Notch 1 mutation

by gene sequencing. Notch 1 gene mutation may result in

overexpression of Notch 1 protein [17]. Immuohisto-

chemical detection of Notch 1 protein is a very common

and simple method in clinical practice. Notch 1 is a trans-

membrane bound receptor expressed in the cytomem-

brane and cytoplasm. Notch 1 antibody, which targets the

Notch 1 protein, can be used to detect expression of

Notch 1 by immunohistochemistry. Franziska identified

Notch 1 protein expression in 90 cases of lymphoma by

immunohistochemistry and found a 100% positivity rate

in classic Hodgkin lymphoma and anaplastic large cell

lymphoma but no expression in B cell lymphoma [18].

Kamstrup also detected the Notch1 expression in cuta-

neous T-cell lymphoma by using immunohistochemistry

and found the prominent expression in 21/40 cases, the

expression of Notch increased with the more advanced

stage [19]. In the present study we found high expression

of Notch l in 68% of T-LBL and low expression in 100%

of B-LBL. Although we did not evaluate for Notch 1

gene mutation in our patients, our finding of high Notch

1 protein expression in T-LBL is similar to the results of

Notch 1 mutation analysis in many studies reporting mu-

tations in more than 50% of T-ALL/LBL cases [7-10].

These results confirm that Notch l may play an important

role in the germination and proliferation of T-LBL but

not B-LBL. Detection of Notch 1 expression by immu-

nohistochemistry may be supplementary to understand-

ing deregulated Notch 1 signaling in T-LBL.

We analyzed the relationship between Notch 1 expres-

sion and clinical features in patients with T-LBL and

found that Notch l expression positively correlated with

the presence of a mediastinal mass. Most T-LBL patients

with an anterior mediastinal mass showed high expres-

sion of Notch 1. Zhu [20] reported that Notch l mutations

were significantly associated with higher blast counts in

the peripheral blood at diagnosis in T-ALL. In spite of

the similarities between T-ALL and T-LBL, there are

also obvious differences in their clinical features. It is

interesting that the predominant manifestation in T-LBL

patients is an anterior mediastinal mass, while in patients

with T-ALL bone marrow involvement is the predomi-

nant site of disease. Thus both findings, i.e. Notch 1 mu-

tation correlating with higher blast counts in the periph-

eral blood in T-ALL and with mediastinal mass in T-

LBL, show that Notch 1 signaling may play an important

role in development of T-ALL/T-LBL.

Multiple clinical series confirm that Notch 1 mutations

in T-ALL/LBL are frequent in all genetic and clinical

subtypes of human T-ALL/LBL, leading investigators to

ask if mutational status is a useful biomarker. However,

associations between mutation status and outcome are in-

consistent. While a few series suggest that Notch 1 muta-

tions are associated with worse outcomes [20], some

studies have shown no association [21,22], and the BFM

groups studies reported that Notch 1 mutations correlate

with better treatment response and overall survival in

T-ALL [8,10]. Callens reported that Notch 1 and/or

FBXW7 mutations were associated with improved event-

free survival and overall survival in pediatric T-lympho-

blastic lymphoma, patients with NOTCH1 mutation de-

monstrated a significantly better outcome than NOTCH1

wild type patients with 96% (SE, 0.04) versus 53% (SE,

Notch 1 and NF-κB Expression and Clinical Correlation in Chinese Patients with Lymphoblastic Lymphoma 445

Table 4. Notch 1 and NF-κB expression and clinical characteristics in T-LBL.

Notch 1 Expression NF-κB Expression

Low High

p value Low High

p value

Male 22 4 (18%) 18 (82%) 4 (18%) 18 (82%)

Sex Female 3 0 (0%) 3 (75%) 0.420 0 (0%) 3 (100%) 0.420

≤18 21 4 (23%) 17 (77%) 4 (18%) 17 (82%)

Age >18 4 0 (0%) 4 (100.0%) 0.341 0 (0%) 4 (100%) 0.341

No 5 3 (60%) 2 (40%) 0 (0%) 5 (100%)

Mediastinal

mass Yes 20 1 (5%) 19 (95%) 0.046 4 (20%) 16 (80%) 0.275

Normal 8 0 (0%) 8 (100%) 2 (25%) 6 (75%)

LDH High 17 4 (23%) 13 (77%) 0.134 2 (12%) 15 (88%) 0.400

0 20 4 (20%) 16 (80%) 4 (20%) 16 (80%)

Extralymph node ≥1 5 0 (0%) 5 (100%) 0.275 0 (0%) 5 (100%) 0.275

III 11 2 (18%) 9 (82%) 4 (36%) 7 (64%)

Stage IV 13 5 (39%) 8 (62%)

0.386 3 (23%) 10 (77%) 0.659

No 20 4 (20%) 16 (80%) 0 (0%) 20 (100%)

B symptoms Yes 5 0 (0%) 5 (100%) 0.275 4 (80%) 1 (20%) 0.000

No 17 2 (12%) 15 (88%) 0 (0%) 17 (100%)

Bulky Yes 8 2 (25%) 6 (75%) 0.088 4 (50%) 4 (50%) 0.013

Figure 3. Survival curve and expression of Notch 1 in T-

LBL.

Figure 4. Survival curve and expression of NF-κB in T-

LBL.

0.11) 5-year EFS, respectively, (p = 0.002) [23], all these

patients received the BFM protocol treatment. It showed

the Notch1 mutation status were associated with survival

in T-ALL/LBL patients who received BFM protocol. So

far the BFM protocol is one of best protocols in treat-

ment of pediatric ALL/LBL. In our study, we only de-

tected the expression of Notch 1 in T-LBL, no detecting

the Notch1 mutation, and found that Notch l expression

did not relate to 5-year event free survival in T-LBL.

Through high expression of Notch1 were found in our

patients, but the sample was small and it did not get a

well evaluation when comparing the association between

the expression and survival. Moreover, the relationship

between Notch l mutation and expression needs to fur-

ther study.

Notch-related T-ALL/LBL is also associated with con-

stitutive activation of NF-κB [24,25]. NF-κB is one of

the important nuclear transcript regulating factors and

plays a role in the onset of T-ALL as a downstream re-

gulator of Notch 1. Blocking this pathway can suppress

carcinoma growth, implying that NF-κB may be a future

treatment target in T-ALL [26]. To produce these thera-

peutic agents, the relationship between NF-κB and Notch

1 expression in LBL should be understood. Up to now,

only a few reports on the expression of NF-κB in LBL

exist. In our research, we found that high expression of

NF-κB occurred in 65% of all LBL cases. There was no

difference in expression of NF-κB between T-LBL and

B-LBL. High expression of NF-κB was significantly re-

lated to bulky disease and B symptom. These findings

show that NF-κB is highly activated in LBL and closely

related to the germination and proliferation of LBL. We

Notch 1 and NF-κB Expression and Clinical Correlation in Chinese Patients with Lymphoblastic Lymphoma

446

analyzed the relationship between expression of NF-κB

and Notch 1 in LBL and found there was a significant

positive correlation between the expression of Notch 1

and NF-κB in T-LBL (p = 0.014). Our results showed

that high expression Notch 1 was accompanied by high

expression of NF-κB in T-LBL, and both have well cor-

relation. This supports the hypothesis that the Notch 1

signaling pathway is associated with constitutive activa-

tion of NF-κB and demonstrates that NF-κB may be a

signal transduction factor downstream of the Notch 1

pathway active in the germination of T-LBL. In contrast,

we found low expression of Notch1 and high expression

of NF-κB in B-LBL, and no correlation between the ex-

pression of Notch 1 and NF-κB in B-LBL, It suggesting

that NF-κB might be activated via other pathways in B-

LBL to result in tumor proliferation.

When analyzing the relationship between NF-κB ex-

pression and clinical characteristics of T-LBL, we found

NF-κB expression to be higher in T-LBL patients with

bulky disease and B symptoms. On the other hand, no

correlation was found between NF-κB expression and

sex, age, or extranodal disease. Bavi detected expression

of NF-κB in 203 cases of diffuse large B cell lymphoma

(DLBCL) using immunohistochemistry and reported that

NF-κB expression occurred in 25.6% (52/203) of DL-

BCL tumors, was associated with activated B cell (ABC)

phenotype, and showed a significantly poorer overall

survival as compared to those without NF-κB expression

[27]. A study in laryngeal squamous cell carcinoma also

showed that overexpression of NF-κB was associated

with worse overall survival and was an independent prog-

nostic factor [28]. In our study we found that NF-κB ex-

pression did not correlate with 5-year event free survival

in T-LBL. However, our sample size was small and fur-

ther investigation is warranted.

This study showed that Notch 1 is highly expressed in

T-LBL and weakly expression in B-LBL. NF-κB was

highly expressed in LBL with no difference between T-

LBL and B-LBL. Notch 1 expression was significantly

associated with NF-κB expression in T-LBL. Notch l and

NF-κB may play important roles in the germination and

development of T-LBL and are potential therapeutic tar-

gets worthy of additional investigation.

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