Introduction
In the Japanese classification, gastric carcinoma is generally divided into two pathological
types, the differentiated type and the undifferentiated type, according to the presence
of histological tubular structures [1]; these two pathological types correspond closely to the intestinal type and diffuse
type of gastric carcinoma, respectively, in Lauren’s classification [2]. Determination of carcinoma differentiation is considered to play a significant
role in the treatment strategy; this is because, in the case of the undifferentiated
type of gastric carcinoma, there is a higher possibility of lymph node metastasis
than is the case for the differentiated type [3].
Several histopathological studies have suggested a correlation between carcinoma differentiation
and intramucosal vascularity. The vascularity of the differentiated type carcinoma
is generally higher than that of the undifferentiated type [4]. This differentiation of vascularity has been reported to influence factors such
as the color of carcinoma observed using conventional endoscopy [5].
Narrow-band imaging (NBI) allows endoscopic observation of minute microsurface structure
or microvessels in the gastric mucosa. In recent years, several studies have reported
the advantages of magnifying endoscopy with NBI (ME-NBI) for Helicobacter pylori induced gastritis: the identification of small gastric tumors; differentiation of
gastric carcinoma from gastritis; and delineation of the horizontal extent of gastric
carcinoma before endoscopic treatment [6]
[7]
[8]
[9]
[10]. Nakayoshi et al. [11] reported that the “network pattern” is often observed in the differentiated type
of gastric carcinoma; however, the “corkscrew pattern” is often observed in the undifferentiated
type of gastric carcinoma. Although the form of the microvessels has been investigated
with regard to the pathological differentiation of gastric carcinoma, microvascular
density using ME-NBI observation has not been investigated in detail.
In the present study, we investigated the differences in characteristics of the density
of microvessels observed using ME-NBI between the differentiated and undifferentiated
types of gastric carcinoma. We also evaluated the histological vascular density using
resected specimens.
Materials and methods
This observational study was performed in a single endoscopy unit at a city hospital
(Sendai City Hospital, Sendai, Miyagi, Japan); the study was conducted in accordance
with the provisions of the Helsinki Declaration. Between April 2010 and July 2011,
74 consecutive patients with intramucosal or submucosal gastric carcinoma who underwent
endoscopic resection or a surgical operation were enrolled. Before carcinoma treatment,
the microvessels were observed using ME-NBI and the images stored in a digital filing
system. The density of the microvessels was evaluated using the stored still images,
and the microvascular density in the two carcinoma types was investigated. A total
of 22 patients were excluded from the study because of inappropriate NBI images as
a result of bleeding, ulceration, or excessive mucosal congestion. Patients with severe
anemia or portal hypertension were also excluded. Patients diagnosed as having a mixture
of the differentiated and undifferentiated types of gastric carcinoma were also excluded
to eliminate complications. Finally, 52 patients were analyzed (42 had differentiated
type and 10 had undifferentiated type carcinoma) in the study. All participating patients
were supplied with a detailed explanation of the study protocol and provided written
informed consent.
Endoscopic procedure
The endoscopic procedure was performed using a CV-260 video system (OLYMPUS Co., Tokyo,
Japan) and a magnifying endoscope (Model H260Z; OLYMPUS). To obtain a clear view using
ME-NBI, a black rubber attachment (MB-162; OLYMPUS) was fitted to the tip of the videoendoscope
to ensure an appropriate distance between the lens and the mucosal surface. A single
experienced endoscopist (M.K.) performed the endoscopic procedure. Before treatment
of the carcinoma, the videoendoscope was inserted into the stomach, and the gastric
disease was visualized using standard and magnifying views. ME-NBI observations were
undertaken approaching the maximum range, and the images (approximately 10 – 50 images)
were saved in a digital filing system. If there were several types of microvessel
in the carcinoma, we analyzed the area with major microvascular forms in the lesion.
The uninvolved mucosa surrounding the carcinoma was also examined using ME-NBI.
Microvessel assessment using ME-NBI
From the stored still ME-NBI images, we extracted microvessels by computer image processing
using image-processing software (ImageJ, version 1.50d; Rasband WS. National Institutes
of Health, Bethesda, Maryland, USA; http://imagej.nih.gov/ij/, 1997 – 2012). We split
the original NBI image in the RGB channel and used the blue color channel to visualize
the microvessels with higher contrast. Using the image, we visually determined the
threshold value for extracting the microvessels. After binarization, we extracted
the vessel area and calculated the ratio of microvessels in the field. We also carried
out this process in the surrounding non-carcinomatous area and calculated the density
of the microvessels. The ratio of ME-NBI assessed vascularity in the carcinoma (C)
to that of the surrounding non-carcinomatous mucosa (N) was also analyzed in each
case.
Histopathological assessment
The resected carcinoma specimens were fixed in buffered formalin and embedded in paraffin.
After hematoxylin and eosin staining, two expert pathologists (H.N. and R.S.) who
were blinded to the ME-NBI findings, assessed the specimens with regard to the differentiation
of gastric carcinoma. Gastric carcinomas were classified as the differentiated type
(with the presence of tubular structures), and the undifferentiated type (with the
absence of tubular structures) in accordance with the Japanese Classification of Gastric
Carcinoma [1].
We determined the histological site in relation to the stored ME-NBI image and assessed
the intramucosal vessels using CD34 immunostaining. Using the stained specimens, we
captured macroscopic images and extracted the vascular field from the image using
computerized and manual techniques. Finally, we calculated the area of vascularity
in both the carcinoma and the surrounding non-carcinomatous areas in the same manner
as that used in the ME-NBI analysis. Because several studies have reported that microvessels
observed using ME-NBI are related to the shallow area of the intramucosal layer [10], we analyzed the histological vascular density not only in the whole mucosal layer
but also in the superficial mucosal layer (0 – 100 µm).
Statistical analysis
Quantitative results are presented as the mean ± standard deviation. The clinical
characteristics were evaluated using the Student’s t test, Fisher’s exact test or the chi-squared test with the Yates continuity correction.
The Student’s unpaired t test was used for the evaluation of microvascular density using ME-NBI and histologically
assessed vascular density. Pearson’s correlation coefficient (r) was used to study the correlations between ME-NBI assessed microvascular density
and histologically assessed vascular density. The Mann–Whitney U test was used for C/N ratio analysis with regard to ME-NBI assessed microvascular
density. Statistical analyses were performed using SPSS Statistics software version
20.0 (SPSS Inc., Chicago, Illinois, United States), with P values of < 0.05 being considered to be statistically significant.
Results
The characteristics of the patients are detailed in [Table 1]. The color of the carcinomas appeared mainly reddish in the differentiated type
and discolored in the undifferentiated type ([Fig. 1] and [Fig. 2]). The ME-NBI assessed microvascular density was 10.02 ± 4.72 % in the differentiated
type and 4.02 ± 0.40 % in the undifferentiated type. There was a statistically significant
difference in ME-NBI assessed microvascular density between the differentiated and
undifferentiated types of carcinoma (P < 0.001; [Table 2]).
Table 1
Characteristics of the patients enrolled in the study.
|
Differentiated type
n = 42
|
Undifferentiated type
n = 10
|
P value
|
|
Age, mean ± SD, years
|
73.3 ± 9.0
|
60.3 ± 10.4
|
< 0.001
|
|
Sex, male/female, n (%)
|
35 (83.3) : 7 (16.7)
|
5 (50.0):5 (50.0)
|
0.039
|
|
Size of tumor mean ± SD, mm
|
18.5 ± 9.8
|
12.1 ± 5.5
|
0.053
|
|
Macroscopic type, 0-IIa/0-IIb/0-IIc, n (%)
|
12 (28.6) : 4 (9.5) : 26 (61.9)
|
0 (0) : 1 (10.0) : 9 (90.0)
|
0.149
|
|
Location, upper/middle/lower, n (%)
|
5 (11.9) : 17 (40.5) : 20 (47.6)
|
1 (10.0) : 8 (80.0) : 1 (10.0)
|
0.065
|
|
Depth of invasion, pT1 m/pT1sm, n (%)
|
34 (81.0) : 8 (19.0)
|
9 (90.0) : 1 (10.0)
|
0.830
|
|
WOS, positive/negative, n (%)
|
8 (19.0) : 34 (81.0)
|
0 (0) : 10 (10.0)
|
0.311
|
|
Color of tumor, reddish/discolored, n (%)
|
31 (73.8) : 11 (26.2)
|
1 (10.0) : 9 (90.0)
|
< 0.001
|
SD, standard deviation; 0-IIa, superficial elevated; 0-IIb, superficial flat; 0-IIc,
superficial depressed; pT1 m, tumor confined to the mucosa; pT1sm, tumor invading
the layer of the submucosa; WOS, white opaque substance.
Fig. 1 a Reddish differentiated type gastric carcinoma in the antrum. b Magnifying endoscopy with narrow-band imaging (ME-NBI) (white box in a indicates location of b; white dashed line: demarcation line of the carcinoma). c Microvessels extracted from the ME-NBI image. NBI vascular density was 11.23 % in
the carcinoma and 3.61 % in the surrounding uninvolved mucosa (white dashed line:
demarcation line of the carcinoma). d Photomicrograph showing a CD34 immunostained tumor tissue section. e Microvessels in the intramucosal layer were extracted. The histologically assessed
vascular density for the carcinoma was 8.79 % in the whole mucosal layer. f The histologically assessed vascular density was 9.26 % in the superficial mucosal
layer (0 – 100 μm). g Photomicrograph showing a CD34 immunostained tissue section from the surrounding
uninvolved mucosa. h The histologically assessed vascular density was 2.75 % in the whole mucosal layer.
I The histologically assessed vascular density was 2.18 % in the superficial mucosal
layer (0 – 100 μm).
Fig. 2 a Discolored undifferentiated type gastric carcinoma in the middle corpus. b Microvessels were extracted from the ME-NBI image (white box in a indicates location of b). The NBI vascular density was 4.34 % in the carcinoma. c The NBI vascular density was 10.59 % in the surrounding uninvolved mucosa (black
box in a indicates location of c). d Photomicrograph showing a CD34 immunostained tissue section from a carcinoma. e The histologically assessed vascular index was 1.49 % in the whole mucosal layer.
f The histologically assessed vascular index was 2.56 % in the superficial mucosal
layer (0 – 100 μm). g Photomicrograph showing a CD34 immunostained tissue section from the surrounding
uninvolved mucosa. h The histologically assessed vascular density was 5.84 % in the whole mucosal layer.
I The histologically assessed vascular density was 6.87 % in the superficial mucosal
layer (0 – 100 μm).
Table 2
Narrow-band imaging (NBI) assessment of microvascular density and histological assessment
of vascular density in gastric carcinoma.
|
Differentiated type carcinoma
|
Undifferentiated type carcinoma
|
P value
|
|
NBI microvascular density, %
|
10.02 ± 4.72
|
4.02 ± 0.40
|
< 0.001
|
|
Histological vascular density, %
|
|
|
|
|
Whole mucosal layer
|
5.81 ± 3.17
|
3.25 ± 1.21
|
0.016
|
|
Surficial mucosal layer (0 – 100 μm)
|
6.38 ± 3.73
|
3.66 ± 1.46
|
0.029
|
The histologically assessed vascular density of the differentiated type carcinoma
was 5.81 ± 3.17 % in the whole mucosal layer and 6.38 ± 3.73 % in the superficial
mucosal layer. The vascular densities of the undifferentiated type carcinoma in the
whole mucosal and superficial mucosal layers were 3.25 ± 1.21 % and 3.66 ± 1.46 %,
respectively. Both in the whole and superficial mucosal layers, the histologically
assessed vascular density in the differentiated type was significantly higher than
that in the undifferentiated type (whole mucosal layer, P = 0.016; superficial mucosal layer, P = 0.029). However, with regard to the surrounding non-carcinomatous mucosa, both
the ME-NBI and the histologically assessed vascular density of the non-carcinomatous
mucosa surrounding the differentiated type carcinoma were significantly lower than
the non-carcinomatous mucosa surrounding the undifferentiated type carcinoma (P < 0.001; [Table 3]). There was a close relationship between ME-NBI assessed microvascular density and
histologically assessed vascular density in both the whole and superficial mucosal
layer (ME-NBI assessed whole mucosal layer, r = 0.740, P < 0.001; ME-NBI assessed superficial mucosal layer, r = 0.764, P < 0.001). The C/N ratio with regard to the ME-NBI assessed vascular density was significantly
higher for the differentiated type carcinoma than for the undifferentiated type carcinoma
(P < 0.001; [Table 4]). White opaque substance (WOS) was seen in eight patients who had the differentiated
type carcinoma ([Fig. 3]); however, WOS was not seen in patients with the undifferentiated type carcinoma.
In almost all cases with WOS (87.5 %), the appearance of the carcinoma was discolored.
Table 3
Narrow-band imaging (NBI) assessment of microvascular density and histological assessment
of vascular density in the surrounding non-carcinomatous mucosa.
|
Non-carcinomatous mucosa surrounding differentiated type carcinoma
|
Non-carcinomatous mucosa surrounding undifferentiated type carcinoma
|
P value
|
|
NBI microvascular density, %
|
5.82 ± 3.39
|
10.99 ± 3.94
|
< 0.001
|
|
Histological vascular density, %
|
|
|
|
|
Whole mucosal layer
|
3.10 ± 1.27
|
7.40 ± 2.51
|
< 0.001
|
|
Surficial mucosal layer (0 – 100 μm)
|
2.49 ± 1.11
|
6.68 ± 3.68
|
< 0.001
|
Table 4
C/N ratio for the microvascular density in the differentiated and undifferentiated
types of carcinoma using NBI.
|
Differentiated type carcinoma
|
Undifferentiated type carcinoma
|
P value
|
|
C/N ratio of NBI vascular density
|
3.94 ± 8.22
|
0.43 ± 0.19
|
< 0.001
|
C, carcinoma; N, non-carcinomatous mucosa; NBI, narrow-band imaging.
Fig. 3 a A discolored differentiated type gastric carcinoma in the antrum. b ME-NBI image showing white opaque substance in the carcinoma, obstructing the visualization
of the microvessels (white box in a indicates location of b).
Discussion
To determine the treatment strategy for early gastric carcinoma, the histopathological
differentiation of gastric carcinoma should be carefully considered. Endoscopic mucosal
resection (EMR) is widely applied for minimally invasive treatment of early gastric
carcinoma; however, undifferentiated type early gastric carcinoma is not generally
considered to be an indication for EMR, even using advanced endoscopic submucosal
dissection techniques. The rate of lymph node metastasis has been demonstrated to
be higher in intramucosal gastric carcinoma of the undifferentiated type (4.2 %) than
that in the differentiated type carcinoma (0.4 %) [3]. Thus, for those patients with a high risk factor concerning the development of
lymph node metastasis, surgical gastrectomy with lymph node dissection should be indicated.
Regarding the determination of the pathological differentiation of gastric carcinoma,
the vascularity of gastric carcinoma within the mucosa has been shown to differ in
previous studies. Adachi et al. [4] examined the mucosal microvascular architecture of early gastric carcinoma using
the Microfil injection method. They demonstrated that the diameter, length, and surface
area of mucosal microvessels in differentiated type carcinoma was significantly higher
than in undifferentiated type carcinoma. They also analyzed microvessels in the surrounding
non-carcinomatous mucosa, and demonstrated that differentiated type carcinomas were
mostly hypervascular or normovascular relative to the surrounding normal mucosa, while
the undifferentiated type carcinomas were often hypovascular. This vascular differentiation
in gastric carcinoma is considered to be related to the progression of tumor growth.
The differentiated type carcinoma was accompanied by the proliferation of vessels
within the neoplastic tissue, although the tumor cells in the undifferentiated type
carcinoma exhibited sparse infiltration with destruction of the normal mucosal vascular
architecture.
Clinically, the difference in mucosal vascularity influences the color change of gastric
carcinoma viewed endoscopically [5]. Differentiated type gastric carcinomas often have a reddish color because of increased
mucosal vascularity, while undifferentiated type gastric carcinomas often appear discolored
because of decreased mucosal vascularity. The present study is the first to reveal
that there was a difference between the microvascular density in the differentiated
and undifferentiated types of gastric carcinoma assessed using ME-NBI. The results
agree with those of previous histopathological vascularity analyses. Our findings
have greater clinical relevance concerning the direct evaluation of microvessels compared
with other studies such as those involving the Hb index calculation [12].
Using spectrally narrow band filters for selecting wavelengths that were absorbed
well by hemoglobin, ME-NBI provides good visualization of microvessels in the superficial
gastric mucosa [13]
[14]. In a previous report [11], the forms of microvessels observed using ME-NBI were useful markers with regard
to the diagnosis of carcinoma differentiation. Our results revealed that not only
the form but also the microvessel densities were useful markers. Our results indicate
that it is necessary to more pay attention to lower density areas of ME-NBI detected
microvessels.
In the present study, we investigated the whole mucosal vascular density histopathologically
using CD34 immunostaining, and demonstrated good agreement with ME-NBI assessed microvascular
density. Several histopathological studies involving mucosal vascularity in gastric
carcinoma have evaluated intramucosal vascular density, using the average microvascular
diameter of selected hot spot areas in the microscopic field. This calculation method
is considered insufficient for the evaluation of whole mucosal blood flow, because
vascularity consists of not only vessel diameter but also length or number of vessels.
Selection of a hot spot of intramucosal vessels in the specimens is also inadequate
for evaluating mucosal vascularity because of limited field size. We believe that
our method is sufficiently robust in evaluating the relationship between endoscopically
assessed microvascular density and histologically assessed vascularity; however, we
should consider the fact that these histopathological images (vertical view) were
not equivalent to the endoscopic images (horizontal view) as a limitation of the study.
In recent studies involving ME-NBI, WOS was sometimes seen in the superficial area
of gastric neoplasia [15]
[16]. WOS that is related to the accumulation of tiny lipid droplets, obstructs the visualization
of the microvessels. In our study, WOS was seen in eight patients who all had the
differentiated type carcinoma; however, WOS was not seen in patients with the undifferentiated
type carcinoma. In almost all cases with WOS, the appearance of the carcinoma was
discolored; thus, WOS is an important factor influencing the color of the carcinoma.
Our methods were inadequate for the investigation of carcinomas with WOS, because
it was impossible to make a diagnosis from the microvascular density or microvascular
using ME-NBI.
A limitation of our study was the small number of patients enrolled, especially those
with undifferentiated type carcinomas. The lack of analysis of mixed histological
types was also an additional limitation. We revealed a significant difference in the
ME-NBI assessed vascular density between differentiated and undifferentiated types
of gastric carcinoma; however, further study is required to determine whether they
have value in the selection of treatment strategies, especially in comparison with
the “gold-standard” biopsy diagnosis. The present study was not blinded; thus, ME-NBI
observation was mainly performed after the diagnosis of histological type using the
biopsy procedure. A prospective multicenter study involving the appropriate number
of ME-NBI images will be required to confirm whether our results are applicable to
daily clinical practice.
In conclusion, we demonstrated a good correlation between ME-NBI and pathologically
assessed vascular density in the superficial and whole mucosal layers. We also revealed
a significant difference in the ME-NBI assessed vascular density between differentiated
and undifferentiated types of gastric carcinoma.
