- Email: [email protected]
-D and lymphangiogenesis in salivary adenoid cystic carcinoma
Accepted Manuscript Title: Expression of VEGF-C/–D and lymphangiogenesis in salivary adenoid cystic carcinoma Author: Ivo St´arek Richard Salzman Ladislava Kuˇcerov´a Alena Sk´alov´a Luk´asˇ Hauer PII: DOI: Reference:
S0344-0338(15)00141-7 http://dx.doi.org/doi:10.1016/j.prp.2015.07.001 PRP 51419
To appear in: Received date: Revised date: Accepted date:
28-1-2015 8-5-2015 3-7-2015
Please cite this article as: I. St´arek, R. Salzman, L. Kuˇcerov´a, A. Sk´alov´a, L. Hauer, Expression of VEGF-C/ndashD and lymphangiogenesis in salivary adenoid cystic carcinoma, Pathology - Research and Practice (2015), http://dx.doi.org/10.1016/j.prp.2015.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Title: Expression of VEGF-C/–D and lymphangiogenesis in salivary adenoid cystic carcinoma Ivo Stárek a #, Richard Salzman a #, Ladislava Kučerová b, Alena Skálová c Lukáš Hauer d a
Clinic of Otorhinolaryngology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky
University Olomouc, Olomouc, Czech Republic b
Department of Clinical and Molecular Pathology, University Hospital Olomouc and Faculty of Medicine and
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Department of Pathology, Charles University in Prague, Faculty of Medicine in Pilsen, Pilsen, Czech Republic Department of Stomatology, Charles University in Prague, Faculty of Medicine in Pilsen, Pilsen, Czech
Republic
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# The first 2 authors contributed equally to the preparation of the manuscript.
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c
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Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
Running title: VEGF in salivary adenoid cystic carcinoma
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Corresponding author: Richard Salzman, Clinic of Otorhinolaryngology, University Hospital Olomouc, Palacký University Olomouc, I. P. Pavlova 6, 775 20, Olomouc, Czech Republic, Tel.: +420 588444186, E-Mail:
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[email protected]
Conflict of Interest Statement: The authors declare that they have no conflict of interest.
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Abbreviations: CR – complete remission, iLVD – intratumoral lymphatic vessel density, LVD – lymphatic
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vessel density, pLVD – peritumoral lymphatic vessel density, SACC – salivary adenoid cystic carcinoma, VEGF-C – vascular endothelial growth factor C, VEGF-D – vascular endothelial growth factor D
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2 Abstract: Aims: Some human neoplasms stimulate lymphangiogenesis through the over-production of vascular endothelial growth factors C/D (VEGF-C/D). Previously little attention has been paid to the mechanisms of lymphogenous spread of salivary adenoid cystic carcinoma (SACC). The current study investigates the presence of lymphatic
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network and the role of VEGF-C and VEGF-D in its formation. Methods: The retrospective study was performed in 20 (12 females and 8 males) patients diagnosed with
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SACC. For the evaluation of VEGF-C/D immunoreactivity, semiquantitative histoscore was calculated as a sum of positive tumor cell score (range 0 to 3) and staining intensity (range 0 to 3). Lymphatic vessel density (LVD)
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was determined as the number of D2-40 positive lymphatic capillaries present at ”hot spots‟. Moreover, the values of histoscores were calculated in surrounding normal parotid parenchyma and compared to those counted
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in tumors. LVD in the tumor center (iLVD), in its periphery (pLVD), and in healthy gland were identified. Results: VEGF-C/D expression, iLVD and pLVD were higher in SACC than in normal gland. The VEGF-
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C/D score correlated neither with pLVD nor with iLVD. High iLVD values were associated with poor survival.
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199/200words
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Conclusions: The authors present the first study demonstrating the existence of lymphatic vessels in SACC.
Key-words: adenoid cystic carcinoma, lymphangiogenesis, salivary gland, vascular endothelial growth factor, VEGF
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Introduction Metastasizing to lymph nodes is a crucial step in the dissemination of malignant epithelial tumors, the mechanisms of which has not been elucidated in all details yet [1]. Traditional concept of this event suggests that tumor cells invade the pre-existing lymphatic network. However, recently there is growing experimental and
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clinical evidence, that some human neoplasms stimulate growth of new lymphatic vessels through the overproduction of various lymphangiogenic cytokines [2-7]. The vascular endothelial growth factors C (VEGF-C)
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and D (VEGF-D) play a dominant role in this proces [8,9]. These two factors, along with VEGF-A (initially called VEGF), B, E, F and placental growth factor belong to the family of angiogenic growth agents, which act
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as ligands for tyrosine-kinase receptors VEGFR-1, VEGFR-2 and VEGFR-3. The former two are present on endothelial cells of blood vessels. On the contrary, VEGFR-3 was detected in lymphatic vessels endothelium.
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Activation of these receptors launches intracelullar transduction cascade, resulting in the blockage of apoptosis, proliferation and migration of endothelial cells of correspondent type of vessels [10-12]. VEGF-C also facilitates
of tumor cells through peritumoral lymphatics [1].
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metastasizing by increasing permeability of the lymphatic endothelium. This promotes intravasation and seeding
In clinical studies, the association between VEGF-C and VEGF-D expression and/or lymphatic vessels density
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(LVD), incidence of nodal metastasis and prognosis of various human carcinomas [13-19], including head and neck squamous cell carcinoma [19-28] was demonstrated. However, only little is known about that matter in salivary gland tumors. Teymoortash [29] compared LVD in Warthin´s tumor and pleomorphic adenoma. Significantly higher values in the former histopathologic entity suggested that its oncocytic cells stimulated lymphangiogenesis. The absence of tumor-associated formation of lymphatic channels in pleomorphic adenomas, was confirmed by Soares [30]. Mello [31] found no significant differences in the VEGF-C expression and LVD between salivary carcinomas with high- and low-risk of nodal involvement according to classification of Regis de Brito Santos [32]. Therefore, he concluded that these tumors applied other than VEGF-C associated molecular mechanisms for their lymphatic spread. Despite the fact that adenoid cystic carcinoma represents one of the most common salivary carcinomas, practically no attention has been paid to the mechanisms of its lymphogenous spread. The probable reason for this negligence is the reported low (usually less than 10%) incidence of nodal metastases from this tumour [33].
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4 Up to now, the only study, dealing with this problem was published by Fujita [34], concluding that lymphangiogenesis does not occur in this histopathological entity. The current study investigates the presence of lymphatic network and the role of VEGF-C and VEGF-D in its formation in salivary adenoid cystic carcinoma (SACC). Moreover, association of immunohistoexpression of these lymphangiogenic factors and LVD with prognosis, histopathological and clinical characteristics of that
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tumor was evaluated.
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Methods Clinical data
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The retrospective study was performed on tissue samples of 20 patients (12 females and 8 males) diagnosed with SACC, at the age ranging from 24 to 84 (mean 56.4+/-15.0) years. The samples were retrieved from Tissue
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Archives of participating pathology departments. Fourteen tumors originated from major and 6 from minor salivary glands. Seven, six, three and four were diagnosed at the clinical stage I, II, III and IV, respectively. Six
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patients presented with clinically positive neck nodes (cN+). Eleven and 9 tumors were histologically graded as G2 and G3, respectively, according to Szanto [35]. Sixteen of seventeen surgically treated patients received
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adjuvant radiotherapy alone or in combination with chemotherapy. The remaining patient was treated with
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surgery only. Radiation therapy and concurrent chemoradiotherapy were applied in one case each. One patient with locally very advanced tumor and distant metastasis refused further treatment after excision biopsy was performed. Three out of 6 N+ patients (one patient refused treatment and two received radiation therapy to N+ necks) received curative neck dissections. Three N0 staged cases (with clinically suspected high stage parotid malignancies and pre- or perioperatively unknown diagnosis of SACC) underwent elective neck dissections. Any use of TNM classification in this manuscript refers to pTNM if available. In remaining (mostly referring to cN0), it refers to cTNM.
Complete remission (CR) was achieved in 18 out of 20 patients. The patients who did not reach CR were excluded from survival analysis. The follow-up ranged between 5 and 286,1 months. Median survival was 46,7 months. Clinico-pathological data are summarized in table I. VEGF-C and VEGF-D immunohistochemistry Sections of 4 μm thickness were cut from each archived paraffin embedded tissue sample. The sections were deparaffinized and rehydrated in graded alcohol. Endogenous peroxidase was blocked in hydrogen peroxide for
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5 15 min. The sections were rinsed in distilled water and subsequently with Tris buffer. Anti-VEGF-C and antiVEGF-D (Abcam, 330 Cambridge Science Park, Cambridge, United Kingdom, dilution 1:50 and 1:100, respectively) and the D2-40 anti-podoplanin (Dako Denmark, Produktionsvej 42, Glostrup, Denmark, dilution 1:100) antibodies were employed. Detection was achieved using the EnVision and Dual System HRP. Enzyme activity was visualized with diaminobenzidine tetrahydrochloride, and the sections were counterstained with
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hematoxylin. Positive and negative controls were prepared according to the manufacturer's recommendations. The staining intensity for VEGF-C and VEGF-D in the cytoplasm and/or nuclei of the tumor and normal parotid
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parenchymal cells was graded according to Moriyama [36]. Intensity equal to that of lymphatic endothelial cells in adjacent normal salivary tissue was determined as grade 2. Absent, weaker, and stronger staining were graded
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as 0, 1, and 3, respectively. The percentage of positive tumor cells was scored according to Lim [15] as 0 (0–19 %), 1 (20–39 %), 2 (40–50 %), and 3 (60–100 %). The sum of intensity and percentage scores provided a
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semiquantitative histoscore (ranging from 0 to 6).
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Evaluation of lymphatic vascular density
Lymphatic microvessels in the tumor center, on its periphery, and in healthy salivary parenchyma were identified
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through immunohistochemical staining using the D2-40 antibody. Intratumoral LVD (iLVD) was assessed in all
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tissue samples. LVD in tumor periphery (pLVD) was evaluated only in samples from major salivary glands, where sufficient surrounding normal parenchyma was present. LVD was also quantified in normal parenchyma remote (>1.5cm distant from tumor margin) from the tumor (n=12). For evaluation of both intra- and peritumoral LVD, the area of highest vascularization called hot spots was identified on a ×40 field, and the number of vessels per square millimeter was counted in a ×200 field. VEGF-C and VEGF-D immunoreactivity as well as LVD was evaluated by two experienced pathologists (LK, AS) blinded to the clinical data. In discrepant findings, specimens were reviewed to achieve conformity. Statistical analysis
For comparison of the mean histoscores between groups, the Mann–Whitney U-test was used. Correlations among histoscores, LVD and clinicopathological parameters were evaluated using the Spearman test. The effect of selected parameters on prognosis was analyzed using Kaplan–Meier survival analysis. The patients alive were classified as „censored“, and those who died as „complete responses“. The prognosis of groups was compared
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6 using log rank test. All analyses were performed with STATISTICA v. 10.0 (Statsoft Inc). A p value of <0.05 was considered statistically significant. Results VEGF-C and D in SACC
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Seventeen out of 20 SACC revealed both VEGF-C and D immunoexpression (Fig. 1a, b, c). Solitary positivity of VEGF-D was found in only one patient, while VEGF-C and D stainings were simultaneously negative
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in 2 samples. VEGF-C, respectively VEGF-D, immunoreaction was at least focally present in all cell types
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(i.e. luminal and non-luminal) constituting the tumor architecture. VEGF histoscores are shown in table II. Lymphatic vessels in SACC
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Intratumoral lymphatic vessels could be demonstrated in all tumors. Peritumoral lymphatics were seen in 15 out of 16 lesions (93.8%). The tumour margins in remaining 4 tumours were largely absent and, therefore, pLVD
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could not be assessed.
to be mostly compressed (Fig. 3a,b).
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Peritumoral lymphatic vessels were mostly dilated (Fig. 2) in contrast to intratumoral vessels which were found
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In addition to lymphatic endothelium, D2-40 positive reaction was present in neoplastic non-luminal cells (Fig. 4a,b).
The iLVD and pLVD values didn’t significantly differ from each other (Spearman, p>0.05, R=0.35). The iLVD and pLVD values are shown in table II.
VEGF-C, D and LVD in normal salivary tissue
In normal salivary gland parenchyma, acinar cells revealed very weak (mostly grade 0) both VEGF-C and VEGF-D reaction, moderate staining of ductal cells was considered unspecific. Lymph vessels in the healthy tissue were seen within interlobular tissue accompanying ducts and blood vessels. VEGF histoscores and LVD values are shown in table II. Myoepithelial cells at the periphery of acini and intracalated ducts showed D2-40 positivity (Fig. 4c). Comparison with normal gland
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7 VEGF-C histoscores were significantly higher in SACC than in acinar cells of normal gland (p=0.003). VEGF-D showed similar tendency without significance (p=0.07). Both iLVD and pLVD were significantly higher than LVD in normal gland (p=0.0009 and p=0.0008, respectively) (Fig. 5). Comparison with clinicopathological parameters
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The VEGF C, D and both iLVD and pLVD revealed no correlation with tumor grade, TNM stage (including presence of nodal metastases), and positivity of surgical margins (Spearman, p>0.05). The postoperative
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positivity of surgical margins was not associated with TNM stage and grade (p>0.05).
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Relationship of VEGF and LVD
The VEGF-C and VEGF-D score correlated neither with the pLVD nor with iLVD (Spearman, all p>0,05).
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Prognostic value of VEGF and LVD
The Kaplan-Meier survival analysis showed no association of VEGF-C, D histoscores and pLVD with over all
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Discussion
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survival (p=0.11). High iLVD values (>9) were associated with poor survival (both log rank test, p=0.002).
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Given the strong tendency of SACC to develop blood metastasis [33], it is not suprising that molecular aspects of hematogenous spread of this tumour were extensively investigated. Relatively numerous experimental and clinical studies demonstrated expression of VEGF, which is the key hemangiogenic growth factor, and its association with the formation of new blood capillaries, tumor size, vascular invasion, distant metastasis and survival [37-42]. On the contrary, in a group of 48 patients with SACC, the VEGF had no predictive value for recurrence and prognosis [49]. VEGF was detected also in the majority of other histopathological types of benign and malignant salivary tumors. However, the reported differences in the VEGF expression between these two groups, as well as the association with clinicopathological factors, including lymph nodes metastasis in carcinomas are controversial [15, 43-47]. Experimentally it was demonstrated that VEGF was able to induce active proliferation not only of tumour associated blood, but also lymphatic vessels [48]. However, Lee found no relationship between expression of this factor and lymph node metastasis in SACC [49]. Only single study dealing with VEGF-C and VEGF--D and associated lymphangiogenesis in SACC has been published so far. In Fujita´s [34] series of 29 tumors scarce, small and mostly constricted lymphatic vessels were localized exclusively at the tumor periphery, with their density not exceeding that of the normal salivary gland.
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8 The VEGF-C immunohistoreaction in tumor cells was barely detectable and VEGF-D was completely absent. However, both proteins were clearly detected in ductal and acinar cells of normal parotid. There was no obvious difference in both the VEGF-C and VEGF-D expressions and LVD between the only one tumor with lymph node metastasis and other tumors without nodal involvement. The author, therefore, concluded that lymphangiogenesis didn‘t occur in SACC.
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The results of Fujita’s study [34] markedly differed from our findings in the following aspects: Firstly, we demonstrated that the majority (17 of 20) of SACC revealed concurrently positive VEGF-C and VEGF-D
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immunohistoreactions, with the correspondent histoscores exceeding those determined in the normal salivary gland acini. This indicates that these lymphangiogenic growth factors are produced by the SACC cells. However,
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we didn’t demonstrate any correlation between either VEGF-C or VEGF-D histoscores and LVD. Similar results were published in head and neck squamous cell carcinomas [21,50] and in a group of 75 various salivary
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carcinomas that were tested by Mello [31]. Plausible explanation of the lacking correlation between tumoral VEGF-C and VEGF-D expressions and LVD might be found in the absence of proteolytic processing of these
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proteins, that is necessary for their effective binding to VEGFR-3 receptors of lymphatic endothelium [51]. Alternatively, VEGF family might not be the only agents involved in lymphangiogenesis in salivary carcinomas,
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as various cytokines were described to regulate this process in other tumors [8]. Namely, SACC is known to
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express two additional lymphagiogenic growth factors, i.e. fibroblast growth factor-1 and 2 [52] and platelet derived growth factor-B [53]. However, their true significance for lymphangiogenesis in salivary carcinomas, especially in SACC, has not been investigated so far. Secondly, in our study both peri- and intratumoral lymphatics, were clearly present. Majority of the former were dilated while the latter showed marked compression. This
pattern is explainable by increased interstitial
pressure exerted on thin vascular walls by the proliferating neoplastic cell growth [54]. Whereas the role of peritumoral lymphatics for the spread of cancerous cells is generally recognized [1], the existence of the functional intratumoral channels has been largely debated. Some studies impugned their presence in various tumors [55-59]. On the other hand, intratumoral lymphatics and positive correlation of their density with lymph node metastasis and prognosis was reported in head and neck squamous cell carcinoma [60]. Munoz-Guerra confirmed the link between intratumoral lymphatics and increased risk of locoregional recurrence in early stage oral carcinoma [61]. In supraglottic carcinomas, Baek [50] suggested that both the intratumoral and peritumoral VEGF-C associated lymphatic vessels were crucial for lymphatic metastasizing. Franchi [62] in his series of 52 head and neck carcinomas found that advanced tumors and those with lymph node metastases had significantly
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9 higher iLVD and pLVD. High pLVD was associated with increased risk of nodal lymph node metastasis, but not with the disease-free or overall survival. Soares [63] examined LVD in 20 minimally and 20 widely invasive carcinomas ex pleomorphic adenoma. In the former group, peritumoral lymphatic vessels significantly outnumbered the intratumoral ones. In the latter tumors, the density of intralymphatic vessel was significantly elevated, equalling to pLVD. The author, therefore, suggested that in the advanced phase of tumor progression,
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intratumoral lymphatics represented an additional pathway increasing its propensity to metastasize. Despite relatively small sample size in our study, the high number of intratumoral, but not peritumoral
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lymphatics associated with poor overall-survival seems to suggest the role of the former in dissemination of SACC cells. On the other hand, there was no correlation between both iLVD and pLVD with presence of nodal
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metastases. Definitive diagnosis of neck lymph nodes involvement of SACC metastases is not easy as elective neck dissection is not considered categorical in N0 patients with SACC [33]. In our study, neck dissection
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allowing definitive assessment of neck lymph nodes involvement, was performed in three cases only. Consequently, we can speculate that in some cases intranodal carcinoma foci might have gone undetected with
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both clinical and imaging diagnostics. As it was noticed by Woolger and Triantafyllou [64], the real incidence of lymph node metastasis from this histopathologic entity is higher than generally thought. This is explainable by
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for a long time.
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minimal production of desmoplastic stroma in SACC, keeping the volume of affected nodes at very low level
In our study of SACC, there was no difference between the number of intratumoral and peritumoral lymphatic vessels. However, they both significantly outnumbered those found in the adjacent normal salivary gland. This differs from papers on salivary pleomorphic adenomas, reporting significantly lower iLVD when compared with pLVD and LVD in normal parotid parenchyma [63,65]. Such differences in LVD between benign pleomorphic adenoma and SACC suggests the abilility of the latter malignancy to stimulate the formation of new lymphatics. Our study is the first one demonstrating the presence of lymphatic vessels in SACC. Positive correlation between their high number and poor prognosis implies that they may participate in distant dissemination of cells of that neoplasm. On the other hand, we failed to confirm the prospective association between density of lymphatic channels and nodal metastasis. Due to the retrospective character of the current study, however, the histopathological status of regional lymph node could not be verified adequately. Moreover, the mechanism the cells of the primary tumor reside the nodes with is very intricate, with lymphangiogenesis representing very important but not the solely step in this process. Therefore, further studies will be necessary to elucidate the role
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10 of VEGF-C and -D as well as other candidate lymphangiogenic growth factors and lymphangiogenesis for the lymphatic spread of SACC. Additionally, this could also open up new possibilities for the targeted molecular therapy, improving as yet dismal prognosis of that neoplasm.
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Acknowledgments: This study was supported by the Institutional Support of the Ministry of Health, Czech Republic, Nr. 1RVO-FNOL2014, RVO: 61989592, and the Internal Grant Agency of the Ministry of Health, Czech Republic, Nr. NT13701-4/2012. Mr. George Kumsta is acknowledged for his help with the final English revision. I. Stárek designed the study, performed the research, analyzed results, wrote the manuscript, R. Salzman
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study, analyzed results, L. Hauer performed staining, took microphotographs
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analyzed results, wrote the manuscript, L. Kučerová performed staining, analyzed results, A. Skálová designed
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[40] J. Liang, Y.H. You, K.X. Ouyng, Y.P. Wang, H.W. Zhang, Expression of vascular endothelial growth factor and microvessel density in salivary adenoid cystic carcinoma, Chin. J. Stomatol. Res. 3,1 (2009) 23-26. [41] Q.L. Tang, W.L. Chen, X.Y. Tan, et al., Expression and significance of Cyr61 in distant metastasis cells of human primary salivary adenoid cystic carcinoma, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 112 (2011) 228-236.
[42] S. Kondo, Y. Mukudai, D. Soga, T. Nishida, M. Takigawa, T. Shirota, Differential expression of vascular endothelial growth factor in high- and low-metastasis cell lines of salivary gland adenoid cystic carcinoma, Anticancer Res. 34 (2014) 671-678. [43] P.R. De Faria, R.A. Lima, F.L. Dias, et al., Vascular endothelial growth factor and tymidine phosphorylase expression in salivary gland tumors with distinct metastatic behavior, J. Oral Pathol. Med. 40 (2011) 456-459. [44] A.C. Faur, E. Lazar, M. Cornianu, Vascular endothelial growth factor (VEGF) expression and microvascular density in salivary gland tumors, APMIS 122 (2013) 418-426.
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15 [45] P. Lequerica-Fernández, A. Astudillo, J.C. De Vicente, Expression of vascular endothelial growth factor in salivary gland carcinoma correlates with lymph node metastasis, Anticancer Res. 27 (2007) 3661-3666. [46] K. Ou Yang, J. Liang, Z.Q. Huang, Association of clinicopathologic parameters with the expression of inducible nitric oxide synthase and vascular endothelial growth factor in mucoepidermoid carcinoma, Oral Dis.
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[48] S. Hirakawa, S. Kodama, R. Kunstfeld, K. Kajiya, L.F. Brown, M. Detmar, VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis, J. Exp. Med. 201 (2005) 1089-
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[49] S.K. Lee, M.S. Kwon, Y.S. Lee, et al., Prognostic value of expression of molecular markers in adenoid
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cystic cancer of the salivary gland compares with lymph node metastasis: a retrospective study, World J. Surg.
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Oncol. 10 (2012) 266-280.
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[50] S.K. Baek, K.Y. Jung, S.H. Lee, et al., Prognostic significance of vascular endothelial growth factor-C expression and lymphatic vessel density in supraglottic squamous cell carcinoma, Laryngoscope 119 (2009) 1325-1330.
[51] N.C. Harris, K. Paavonen, N. Davydova, et al., Proteolytic processing of vascular endothelial growth factorD is essential for its capacity to promote growth and spread of cancer, FASEB J. 25,8 (2001) 2615-2625. [52] Y. Myoken, Y. Myoken,T. Okamoto, et al., Immunohistochemical study of overexpression of fibroblast growth factor-1 (FGF-1), FGF-2, and FGF receptor 1 in human malignant salivary gland tumours, J. Pathol. 178 (1996) 429-436.
[53] H. Vekony, B. Ylstra, S.M. Wilting, et al., DNA copy number gains at loci of growth factors and their receptors in salivary gland adenoid cystic carcinoma, Clin. Cancer Res. 13 (2007) 3133-3139. [54] T.P. Padera, B.R. Stoll, J.B. Tooredman, D. Capen, F. di Tomaso, R.K. Jain, Cancer cells compress intratumour vessels, Nature 427 (2004) 695.
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16 [55] N. Tanigawa, T. Kanazawa, K. Satomura, et al., Experimental study on lymphatic vascular changes in the development of cancer, Lymphology 14 (1981) 149-154. [56] M. Xuan, Y.R. Fang, M. Wato, S. Hata, A. Tanaka, Immunohistochemical co-localization of lymphatics and blood vessels in oral squamous cell carcinomas, J. Oral Pathol. Med. 34 (2005) 334-339.
lymphangiogenesis in endometrial and lung cancer, J. Clin. Pathol. 58 (2005) 202-206.
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[57] M.I. Koukourakis, A. Giatromanolaki, E. Sivridis, et al., LYVE-1 immunohistochemical assessemnt of
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vessels in human metastasic breast cancer, J. Pathol. 200 (2003) 195-206.
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[58] C.S.M. Williams, R.D. Leek, A.M. Robson, et al., Absence of lymphangiogensis and intratumoral lymph
[59] B. Agarwal, R. Saxena, A. Morimiya, et al., Lymphangiogensis does not occur in breast cancer, Am. J.
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Pathol. 29 (2005) 1449-1455.
[60] S.M. Maula, M. Luukkaa, R. Grenman, D. Jackson, S. Jalkanen, R. Ristamaki, Intratumoral lymphatics are
Res. 63 (2003) 1920-1926.
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essential for the metastatis spread and prognosis in squamous cell carcinoma of the head and neck region, Cancer
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[61] M.F. Munoz-Guerra, E.G. Marazuela, E. Martin-Villar, M. Quantanilla, C. Gamallo, Prognostic
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significance of intratumoral lymphangiogenesis in squamous cell carcinoma of the oral cavity. A study of the early stages, Cancer 100 (2004) 553-560.
[62] A. Franchi, O. Gallo, D. Massi, G. Baroni, M. Santucci, Tumor lymphangiogenesis in head and neck squamous cell carcinoma. A morphometric study with clinical correlations, Cancer 101,5 (2004) 973-978. [63] A.B. Soares, L. Ponchio, P.B. Juliano, V.C. de Araújo, A. Altemani, Lymphatic vascular density and lymphagiogenesis during tumor progression of carcinoma ex pleomorphic adenoma, J. Clin. Pathol. 60,9 (2007) 995-1000.
[64] J.A. Woolger, A. Triantafullou, Contemporary salivary clinical pathology. Facts and dilemmas, in: P.J. Bradley, O. Guntinas-Lichius (Eds.), Salivary gland disorders and diseases: Diagnosis and management, Thieme Verlag, Stuttgart, 2011, pp. 27-41.
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17 [65] R. Salzman, I. Stárek, L. Kučerová, A. Skálová, J. Hoza, Neither expression of VEGF-C/D nor lymph vessel density supports lymphatic invasion as the mechanism responsible for local spread of recurrent salivary
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pleomorphic adenoma, Virchows Arch 464 (2014) 29-34.
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18 Figure legend Fig. 1 Cribriform structures of SACC (cribriform/tubular variant, grade 2), Fig. 1a) with moderate (2+) VEGFC, Fig. 1b) and strong (3+) VEGF-D, Fig. 1c) cytoplasmic and nuclear immunoreaction.
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Fig. 2 D2-40 positive intratumoral lymphatic vessels Fig. 3a Cribriform structures of SACC (solid/cribriform variant, grade 3), Fig. 3b). Intratumoral lymphatic
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vessels lined with D2-40 positive endothelial cells are completely (arrow) or partially (arrowhead) compresssed. Fig. 4 Cribriform structures of SACC (cribriform/tubular variant, grade 2), Fig. 4a) D2-40 positive
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myoepithelial cells of SACC, Fig. 4b) no intratumoral lymphatics, no staining for D2-40 in blood vessel
intercalated ducts (arrows) of normal salivary parenchyma.
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endothelial cells (asterisk). Fig. 4c) D2-40 positive myoepithelial cells surrounding acini (arrowhead) and
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significant differences using Mann-Whitney test.
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Fig. 5 Lymphatic vessel density in the tumor, in its periphery, and in normal salivary parenchyma show
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19 Tables
Location
Treatment*
SM*
CR*
Outcome* F/U (months)
1
F/57
T3N0M0
parotid
S+ND+RT
neg.
yes
NED
14
2
M/32
T1N0M0
submand.
S+RT
neg.
yes
NED
26
3
M/84
T4N3bM1
buccal gland
no treatment
-
no
DOD
9
4
F/58
T2N0M0
buccal gland
S+RT+CHT
pos.
yes
NED
96
5
F/60
T1N0M0
maxilla
S
neg.
yes
AWD
59
6
F/48
T2N0M0
hard palate
S+RT+CHT
pos.
yes
NED
84
7
M/57
T4N2bM0
parotid
S+ND+RT+CHT
pos.
yes
DOD
18
8
F/48
T3N0M0
submand.
RT+CHT
N/A
yes
DOD
137
9
F/47
T1N1M0
submand.
S+ND+RT+CHT
neg.
yes
DOD
44
10
M/65
T1N0M0
sublingual
S+ND+RT
pos.
yes
NED
80
11
M/45
T3N0M0
parotid
S+ND+RT
pos.
yes
NED
46
12
F/56
T3N0M0
parotid
S+RT
pos.
yes
AWD
172
13
F/79
T4N1M0
palat. tonsil
RT
N/A
no
DOD
7
14
F/77
T2N0M0
parotid
S+RT
neg.
yes
NED
22
16 17 18 19 20
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15
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Sex/Age TNM staging
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Pat.
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Table 1. Clinicopathologic data of patients with SACC
F/49
T3N0M0
submand.
S+RT+CHT
neg.
yes
NED
162
F/76
T3N1M0
sublingual.
S+ND+RT
neg.
yes
NED
5
F/54
T1N0M0
submand.
S+RT
N/A
yes
DOD
130
M/51
T4N2bM0
max. alv. ridge
S+RT
neg.
yes
NED
86
M/24
T1N0M0
submand.
S+RT
neg.
yes
NED
282
M/61
T3N0M0
deep parotid
S+RT
pos.
yes
AWD
7
* Abbr. AWD - alive with disease, CHT - chemotherapy, CR - complete remission, DOD - dead of disease, F/U - follow-up, N/A - not available, ND - neck dissection, NED - no evidence of disease, RT - radiotherapy, S - surgery, SM - surgical margins
Page 19 of 31
20
Parameter*
Min
Max
Mean
SD
VEGF-C
0
5
3.65
1.25
VEGF-D
3
6
3.82
0.88
iLVD
1
15
6.32
3.83
pLVD
0
15
7.0
4.04
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Table 2. VEGF histoscore and LVD in SACC
* Abbr. iLVD - intratumoral lymphatic vessel density, pLVD - peritumoral lymphatic vessel density, SD - standard deviation,
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VEGF-C - vascular endothelial growth factor C, VEGF-D - vascular endothelial growth factor D
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21 Table 3. VEGF histoscores in acinar cells and LVD in normal parenchyma Parameter*
Min
Max
Mean
SD
VEGF-C
0
4
1.67
1.78
VEGF-D
0
5
2.33
1.92
LVD
0
8
1.75
2.56
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* Abbr. LVD - lymphatic vessel density, SD - standard deviation, VEGF-C - vascular endothelial growth factor C, VEGF-D -
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vascular endothelial growth factor D
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pt
ed
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Figure1a
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Figure1b
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Figure1c
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Figure2
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Figure3a
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Figure3b
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Figure4a
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Figure4b
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Figure4c
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Figure5
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S0344-0338(15)00141-7 http://dx.doi.org/doi:10.1016/j.prp.2015.07.001 PRP 51419
To appear in: Received date: Revised date: Accepted date:
28-1-2015 8-5-2015 3-7-2015
Please cite this article as: I. St´arek, R. Salzman, L. Kuˇcerov´a, A. Sk´alov´a, L. Hauer, Expression of VEGF-C/ndashD and lymphangiogenesis in salivary adenoid cystic carcinoma, Pathology - Research and Practice (2015), http://dx.doi.org/10.1016/j.prp.2015.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Title: Expression of VEGF-C/–D and lymphangiogenesis in salivary adenoid cystic carcinoma Ivo Stárek a #, Richard Salzman a #, Ladislava Kučerová b, Alena Skálová c Lukáš Hauer d a
Clinic of Otorhinolaryngology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky
University Olomouc, Olomouc, Czech Republic b
Department of Clinical and Molecular Pathology, University Hospital Olomouc and Faculty of Medicine and
d
Department of Pathology, Charles University in Prague, Faculty of Medicine in Pilsen, Pilsen, Czech Republic Department of Stomatology, Charles University in Prague, Faculty of Medicine in Pilsen, Pilsen, Czech
Republic
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# The first 2 authors contributed equally to the preparation of the manuscript.
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c
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Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
Running title: VEGF in salivary adenoid cystic carcinoma
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Corresponding author: Richard Salzman, Clinic of Otorhinolaryngology, University Hospital Olomouc, Palacký University Olomouc, I. P. Pavlova 6, 775 20, Olomouc, Czech Republic, Tel.: +420 588444186, E-Mail:
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[email protected]
Conflict of Interest Statement: The authors declare that they have no conflict of interest.
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Abbreviations: CR – complete remission, iLVD – intratumoral lymphatic vessel density, LVD – lymphatic
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vessel density, pLVD – peritumoral lymphatic vessel density, SACC – salivary adenoid cystic carcinoma, VEGF-C – vascular endothelial growth factor C, VEGF-D – vascular endothelial growth factor D
Page 1 of 31
2 Abstract: Aims: Some human neoplasms stimulate lymphangiogenesis through the over-production of vascular endothelial growth factors C/D (VEGF-C/D). Previously little attention has been paid to the mechanisms of lymphogenous spread of salivary adenoid cystic carcinoma (SACC). The current study investigates the presence of lymphatic
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network and the role of VEGF-C and VEGF-D in its formation. Methods: The retrospective study was performed in 20 (12 females and 8 males) patients diagnosed with
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SACC. For the evaluation of VEGF-C/D immunoreactivity, semiquantitative histoscore was calculated as a sum of positive tumor cell score (range 0 to 3) and staining intensity (range 0 to 3). Lymphatic vessel density (LVD)
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was determined as the number of D2-40 positive lymphatic capillaries present at ”hot spots‟. Moreover, the values of histoscores were calculated in surrounding normal parotid parenchyma and compared to those counted
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in tumors. LVD in the tumor center (iLVD), in its periphery (pLVD), and in healthy gland were identified. Results: VEGF-C/D expression, iLVD and pLVD were higher in SACC than in normal gland. The VEGF-
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C/D score correlated neither with pLVD nor with iLVD. High iLVD values were associated with poor survival.
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199/200words
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Conclusions: The authors present the first study demonstrating the existence of lymphatic vessels in SACC.
Key-words: adenoid cystic carcinoma, lymphangiogenesis, salivary gland, vascular endothelial growth factor, VEGF
Page 2 of 31
3
Introduction Metastasizing to lymph nodes is a crucial step in the dissemination of malignant epithelial tumors, the mechanisms of which has not been elucidated in all details yet [1]. Traditional concept of this event suggests that tumor cells invade the pre-existing lymphatic network. However, recently there is growing experimental and
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clinical evidence, that some human neoplasms stimulate growth of new lymphatic vessels through the overproduction of various lymphangiogenic cytokines [2-7]. The vascular endothelial growth factors C (VEGF-C)
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and D (VEGF-D) play a dominant role in this proces [8,9]. These two factors, along with VEGF-A (initially called VEGF), B, E, F and placental growth factor belong to the family of angiogenic growth agents, which act
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as ligands for tyrosine-kinase receptors VEGFR-1, VEGFR-2 and VEGFR-3. The former two are present on endothelial cells of blood vessels. On the contrary, VEGFR-3 was detected in lymphatic vessels endothelium.
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Activation of these receptors launches intracelullar transduction cascade, resulting in the blockage of apoptosis, proliferation and migration of endothelial cells of correspondent type of vessels [10-12]. VEGF-C also facilitates
of tumor cells through peritumoral lymphatics [1].
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metastasizing by increasing permeability of the lymphatic endothelium. This promotes intravasation and seeding
In clinical studies, the association between VEGF-C and VEGF-D expression and/or lymphatic vessels density
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(LVD), incidence of nodal metastasis and prognosis of various human carcinomas [13-19], including head and neck squamous cell carcinoma [19-28] was demonstrated. However, only little is known about that matter in salivary gland tumors. Teymoortash [29] compared LVD in Warthin´s tumor and pleomorphic adenoma. Significantly higher values in the former histopathologic entity suggested that its oncocytic cells stimulated lymphangiogenesis. The absence of tumor-associated formation of lymphatic channels in pleomorphic adenomas, was confirmed by Soares [30]. Mello [31] found no significant differences in the VEGF-C expression and LVD between salivary carcinomas with high- and low-risk of nodal involvement according to classification of Regis de Brito Santos [32]. Therefore, he concluded that these tumors applied other than VEGF-C associated molecular mechanisms for their lymphatic spread. Despite the fact that adenoid cystic carcinoma represents one of the most common salivary carcinomas, practically no attention has been paid to the mechanisms of its lymphogenous spread. The probable reason for this negligence is the reported low (usually less than 10%) incidence of nodal metastases from this tumour [33].
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4 Up to now, the only study, dealing with this problem was published by Fujita [34], concluding that lymphangiogenesis does not occur in this histopathological entity. The current study investigates the presence of lymphatic network and the role of VEGF-C and VEGF-D in its formation in salivary adenoid cystic carcinoma (SACC). Moreover, association of immunohistoexpression of these lymphangiogenic factors and LVD with prognosis, histopathological and clinical characteristics of that
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tumor was evaluated.
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Methods Clinical data
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The retrospective study was performed on tissue samples of 20 patients (12 females and 8 males) diagnosed with SACC, at the age ranging from 24 to 84 (mean 56.4+/-15.0) years. The samples were retrieved from Tissue
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Archives of participating pathology departments. Fourteen tumors originated from major and 6 from minor salivary glands. Seven, six, three and four were diagnosed at the clinical stage I, II, III and IV, respectively. Six
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patients presented with clinically positive neck nodes (cN+). Eleven and 9 tumors were histologically graded as G2 and G3, respectively, according to Szanto [35]. Sixteen of seventeen surgically treated patients received
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adjuvant radiotherapy alone or in combination with chemotherapy. The remaining patient was treated with
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surgery only. Radiation therapy and concurrent chemoradiotherapy were applied in one case each. One patient with locally very advanced tumor and distant metastasis refused further treatment after excision biopsy was performed. Three out of 6 N+ patients (one patient refused treatment and two received radiation therapy to N+ necks) received curative neck dissections. Three N0 staged cases (with clinically suspected high stage parotid malignancies and pre- or perioperatively unknown diagnosis of SACC) underwent elective neck dissections. Any use of TNM classification in this manuscript refers to pTNM if available. In remaining (mostly referring to cN0), it refers to cTNM.
Complete remission (CR) was achieved in 18 out of 20 patients. The patients who did not reach CR were excluded from survival analysis. The follow-up ranged between 5 and 286,1 months. Median survival was 46,7 months. Clinico-pathological data are summarized in table I. VEGF-C and VEGF-D immunohistochemistry Sections of 4 μm thickness were cut from each archived paraffin embedded tissue sample. The sections were deparaffinized and rehydrated in graded alcohol. Endogenous peroxidase was blocked in hydrogen peroxide for
Page 4 of 31
5 15 min. The sections were rinsed in distilled water and subsequently with Tris buffer. Anti-VEGF-C and antiVEGF-D (Abcam, 330 Cambridge Science Park, Cambridge, United Kingdom, dilution 1:50 and 1:100, respectively) and the D2-40 anti-podoplanin (Dako Denmark, Produktionsvej 42, Glostrup, Denmark, dilution 1:100) antibodies were employed. Detection was achieved using the EnVision and Dual System HRP. Enzyme activity was visualized with diaminobenzidine tetrahydrochloride, and the sections were counterstained with
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hematoxylin. Positive and negative controls were prepared according to the manufacturer's recommendations. The staining intensity for VEGF-C and VEGF-D in the cytoplasm and/or nuclei of the tumor and normal parotid
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parenchymal cells was graded according to Moriyama [36]. Intensity equal to that of lymphatic endothelial cells in adjacent normal salivary tissue was determined as grade 2. Absent, weaker, and stronger staining were graded
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as 0, 1, and 3, respectively. The percentage of positive tumor cells was scored according to Lim [15] as 0 (0–19 %), 1 (20–39 %), 2 (40–50 %), and 3 (60–100 %). The sum of intensity and percentage scores provided a
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semiquantitative histoscore (ranging from 0 to 6).
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Evaluation of lymphatic vascular density
Lymphatic microvessels in the tumor center, on its periphery, and in healthy salivary parenchyma were identified
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through immunohistochemical staining using the D2-40 antibody. Intratumoral LVD (iLVD) was assessed in all
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tissue samples. LVD in tumor periphery (pLVD) was evaluated only in samples from major salivary glands, where sufficient surrounding normal parenchyma was present. LVD was also quantified in normal parenchyma remote (>1.5cm distant from tumor margin) from the tumor (n=12). For evaluation of both intra- and peritumoral LVD, the area of highest vascularization called hot spots was identified on a ×40 field, and the number of vessels per square millimeter was counted in a ×200 field. VEGF-C and VEGF-D immunoreactivity as well as LVD was evaluated by two experienced pathologists (LK, AS) blinded to the clinical data. In discrepant findings, specimens were reviewed to achieve conformity. Statistical analysis
For comparison of the mean histoscores between groups, the Mann–Whitney U-test was used. Correlations among histoscores, LVD and clinicopathological parameters were evaluated using the Spearman test. The effect of selected parameters on prognosis was analyzed using Kaplan–Meier survival analysis. The patients alive were classified as „censored“, and those who died as „complete responses“. The prognosis of groups was compared
Page 5 of 31
6 using log rank test. All analyses were performed with STATISTICA v. 10.0 (Statsoft Inc). A p value of <0.05 was considered statistically significant. Results VEGF-C and D in SACC
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Seventeen out of 20 SACC revealed both VEGF-C and D immunoexpression (Fig. 1a, b, c). Solitary positivity of VEGF-D was found in only one patient, while VEGF-C and D stainings were simultaneously negative
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in 2 samples. VEGF-C, respectively VEGF-D, immunoreaction was at least focally present in all cell types
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(i.e. luminal and non-luminal) constituting the tumor architecture. VEGF histoscores are shown in table II. Lymphatic vessels in SACC
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Intratumoral lymphatic vessels could be demonstrated in all tumors. Peritumoral lymphatics were seen in 15 out of 16 lesions (93.8%). The tumour margins in remaining 4 tumours were largely absent and, therefore, pLVD
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could not be assessed.
to be mostly compressed (Fig. 3a,b).
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Peritumoral lymphatic vessels were mostly dilated (Fig. 2) in contrast to intratumoral vessels which were found
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In addition to lymphatic endothelium, D2-40 positive reaction was present in neoplastic non-luminal cells (Fig. 4a,b).
The iLVD and pLVD values didn’t significantly differ from each other (Spearman, p>0.05, R=0.35). The iLVD and pLVD values are shown in table II.
VEGF-C, D and LVD in normal salivary tissue
In normal salivary gland parenchyma, acinar cells revealed very weak (mostly grade 0) both VEGF-C and VEGF-D reaction, moderate staining of ductal cells was considered unspecific. Lymph vessels in the healthy tissue were seen within interlobular tissue accompanying ducts and blood vessels. VEGF histoscores and LVD values are shown in table II. Myoepithelial cells at the periphery of acini and intracalated ducts showed D2-40 positivity (Fig. 4c). Comparison with normal gland
Page 6 of 31
7 VEGF-C histoscores were significantly higher in SACC than in acinar cells of normal gland (p=0.003). VEGF-D showed similar tendency without significance (p=0.07). Both iLVD and pLVD were significantly higher than LVD in normal gland (p=0.0009 and p=0.0008, respectively) (Fig. 5). Comparison with clinicopathological parameters
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The VEGF C, D and both iLVD and pLVD revealed no correlation with tumor grade, TNM stage (including presence of nodal metastases), and positivity of surgical margins (Spearman, p>0.05). The postoperative
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positivity of surgical margins was not associated with TNM stage and grade (p>0.05).
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Relationship of VEGF and LVD
The VEGF-C and VEGF-D score correlated neither with the pLVD nor with iLVD (Spearman, all p>0,05).
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Prognostic value of VEGF and LVD
The Kaplan-Meier survival analysis showed no association of VEGF-C, D histoscores and pLVD with over all
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Discussion
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survival (p=0.11). High iLVD values (>9) were associated with poor survival (both log rank test, p=0.002).
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Given the strong tendency of SACC to develop blood metastasis [33], it is not suprising that molecular aspects of hematogenous spread of this tumour were extensively investigated. Relatively numerous experimental and clinical studies demonstrated expression of VEGF, which is the key hemangiogenic growth factor, and its association with the formation of new blood capillaries, tumor size, vascular invasion, distant metastasis and survival [37-42]. On the contrary, in a group of 48 patients with SACC, the VEGF had no predictive value for recurrence and prognosis [49]. VEGF was detected also in the majority of other histopathological types of benign and malignant salivary tumors. However, the reported differences in the VEGF expression between these two groups, as well as the association with clinicopathological factors, including lymph nodes metastasis in carcinomas are controversial [15, 43-47]. Experimentally it was demonstrated that VEGF was able to induce active proliferation not only of tumour associated blood, but also lymphatic vessels [48]. However, Lee found no relationship between expression of this factor and lymph node metastasis in SACC [49]. Only single study dealing with VEGF-C and VEGF--D and associated lymphangiogenesis in SACC has been published so far. In Fujita´s [34] series of 29 tumors scarce, small and mostly constricted lymphatic vessels were localized exclusively at the tumor periphery, with their density not exceeding that of the normal salivary gland.
Page 7 of 31
8 The VEGF-C immunohistoreaction in tumor cells was barely detectable and VEGF-D was completely absent. However, both proteins were clearly detected in ductal and acinar cells of normal parotid. There was no obvious difference in both the VEGF-C and VEGF-D expressions and LVD between the only one tumor with lymph node metastasis and other tumors without nodal involvement. The author, therefore, concluded that lymphangiogenesis didn‘t occur in SACC.
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The results of Fujita’s study [34] markedly differed from our findings in the following aspects: Firstly, we demonstrated that the majority (17 of 20) of SACC revealed concurrently positive VEGF-C and VEGF-D
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immunohistoreactions, with the correspondent histoscores exceeding those determined in the normal salivary gland acini. This indicates that these lymphangiogenic growth factors are produced by the SACC cells. However,
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we didn’t demonstrate any correlation between either VEGF-C or VEGF-D histoscores and LVD. Similar results were published in head and neck squamous cell carcinomas [21,50] and in a group of 75 various salivary
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carcinomas that were tested by Mello [31]. Plausible explanation of the lacking correlation between tumoral VEGF-C and VEGF-D expressions and LVD might be found in the absence of proteolytic processing of these
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proteins, that is necessary for their effective binding to VEGFR-3 receptors of lymphatic endothelium [51]. Alternatively, VEGF family might not be the only agents involved in lymphangiogenesis in salivary carcinomas,
d
as various cytokines were described to regulate this process in other tumors [8]. Namely, SACC is known to
Ac ce pt e
express two additional lymphagiogenic growth factors, i.e. fibroblast growth factor-1 and 2 [52] and platelet derived growth factor-B [53]. However, their true significance for lymphangiogenesis in salivary carcinomas, especially in SACC, has not been investigated so far. Secondly, in our study both peri- and intratumoral lymphatics, were clearly present. Majority of the former were dilated while the latter showed marked compression. This
pattern is explainable by increased interstitial
pressure exerted on thin vascular walls by the proliferating neoplastic cell growth [54]. Whereas the role of peritumoral lymphatics for the spread of cancerous cells is generally recognized [1], the existence of the functional intratumoral channels has been largely debated. Some studies impugned their presence in various tumors [55-59]. On the other hand, intratumoral lymphatics and positive correlation of their density with lymph node metastasis and prognosis was reported in head and neck squamous cell carcinoma [60]. Munoz-Guerra confirmed the link between intratumoral lymphatics and increased risk of locoregional recurrence in early stage oral carcinoma [61]. In supraglottic carcinomas, Baek [50] suggested that both the intratumoral and peritumoral VEGF-C associated lymphatic vessels were crucial for lymphatic metastasizing. Franchi [62] in his series of 52 head and neck carcinomas found that advanced tumors and those with lymph node metastases had significantly
Page 8 of 31
9 higher iLVD and pLVD. High pLVD was associated with increased risk of nodal lymph node metastasis, but not with the disease-free or overall survival. Soares [63] examined LVD in 20 minimally and 20 widely invasive carcinomas ex pleomorphic adenoma. In the former group, peritumoral lymphatic vessels significantly outnumbered the intratumoral ones. In the latter tumors, the density of intralymphatic vessel was significantly elevated, equalling to pLVD. The author, therefore, suggested that in the advanced phase of tumor progression,
ip t
intratumoral lymphatics represented an additional pathway increasing its propensity to metastasize. Despite relatively small sample size in our study, the high number of intratumoral, but not peritumoral
cr
lymphatics associated with poor overall-survival seems to suggest the role of the former in dissemination of SACC cells. On the other hand, there was no correlation between both iLVD and pLVD with presence of nodal
us
metastases. Definitive diagnosis of neck lymph nodes involvement of SACC metastases is not easy as elective neck dissection is not considered categorical in N0 patients with SACC [33]. In our study, neck dissection
an
allowing definitive assessment of neck lymph nodes involvement, was performed in three cases only. Consequently, we can speculate that in some cases intranodal carcinoma foci might have gone undetected with
M
both clinical and imaging diagnostics. As it was noticed by Woolger and Triantafyllou [64], the real incidence of lymph node metastasis from this histopathologic entity is higher than generally thought. This is explainable by
Ac ce pt e
for a long time.
d
minimal production of desmoplastic stroma in SACC, keeping the volume of affected nodes at very low level
In our study of SACC, there was no difference between the number of intratumoral and peritumoral lymphatic vessels. However, they both significantly outnumbered those found in the adjacent normal salivary gland. This differs from papers on salivary pleomorphic adenomas, reporting significantly lower iLVD when compared with pLVD and LVD in normal parotid parenchyma [63,65]. Such differences in LVD between benign pleomorphic adenoma and SACC suggests the abilility of the latter malignancy to stimulate the formation of new lymphatics. Our study is the first one demonstrating the presence of lymphatic vessels in SACC. Positive correlation between their high number and poor prognosis implies that they may participate in distant dissemination of cells of that neoplasm. On the other hand, we failed to confirm the prospective association between density of lymphatic channels and nodal metastasis. Due to the retrospective character of the current study, however, the histopathological status of regional lymph node could not be verified adequately. Moreover, the mechanism the cells of the primary tumor reside the nodes with is very intricate, with lymphangiogenesis representing very important but not the solely step in this process. Therefore, further studies will be necessary to elucidate the role
Page 9 of 31
10 of VEGF-C and -D as well as other candidate lymphangiogenic growth factors and lymphangiogenesis for the lymphatic spread of SACC. Additionally, this could also open up new possibilities for the targeted molecular therapy, improving as yet dismal prognosis of that neoplasm.
ip t
Acknowledgments: This study was supported by the Institutional Support of the Ministry of Health, Czech Republic, Nr. 1RVO-FNOL2014, RVO: 61989592, and the Internal Grant Agency of the Ministry of Health, Czech Republic, Nr. NT13701-4/2012. Mr. George Kumsta is acknowledged for his help with the final English revision. I. Stárek designed the study, performed the research, analyzed results, wrote the manuscript, R. Salzman
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d
M
an
us
study, analyzed results, L. Hauer performed staining, took microphotographs
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analyzed results, wrote the manuscript, L. Kučerová performed staining, analyzed results, A. Skálová designed
Page 10 of 31
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17 [65] R. Salzman, I. Stárek, L. Kučerová, A. Skálová, J. Hoza, Neither expression of VEGF-C/D nor lymph vessel density supports lymphatic invasion as the mechanism responsible for local spread of recurrent salivary
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pleomorphic adenoma, Virchows Arch 464 (2014) 29-34.
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18 Figure legend Fig. 1 Cribriform structures of SACC (cribriform/tubular variant, grade 2), Fig. 1a) with moderate (2+) VEGFC, Fig. 1b) and strong (3+) VEGF-D, Fig. 1c) cytoplasmic and nuclear immunoreaction.
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Fig. 2 D2-40 positive intratumoral lymphatic vessels Fig. 3a Cribriform structures of SACC (solid/cribriform variant, grade 3), Fig. 3b). Intratumoral lymphatic
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vessels lined with D2-40 positive endothelial cells are completely (arrow) or partially (arrowhead) compresssed. Fig. 4 Cribriform structures of SACC (cribriform/tubular variant, grade 2), Fig. 4a) D2-40 positive
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myoepithelial cells of SACC, Fig. 4b) no intratumoral lymphatics, no staining for D2-40 in blood vessel
intercalated ducts (arrows) of normal salivary parenchyma.
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endothelial cells (asterisk). Fig. 4c) D2-40 positive myoepithelial cells surrounding acini (arrowhead) and
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significant differences using Mann-Whitney test.
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Fig. 5 Lymphatic vessel density in the tumor, in its periphery, and in normal salivary parenchyma show
Page 18 of 31
19 Tables
Location
Treatment*
SM*
CR*
Outcome* F/U (months)
1
F/57
T3N0M0
parotid
S+ND+RT
neg.
yes
NED
14
2
M/32
T1N0M0
submand.
S+RT
neg.
yes
NED
26
3
M/84
T4N3bM1
buccal gland
no treatment
-
no
DOD
9
4
F/58
T2N0M0
buccal gland
S+RT+CHT
pos.
yes
NED
96
5
F/60
T1N0M0
maxilla
S
neg.
yes
AWD
59
6
F/48
T2N0M0
hard palate
S+RT+CHT
pos.
yes
NED
84
7
M/57
T4N2bM0
parotid
S+ND+RT+CHT
pos.
yes
DOD
18
8
F/48
T3N0M0
submand.
RT+CHT
N/A
yes
DOD
137
9
F/47
T1N1M0
submand.
S+ND+RT+CHT
neg.
yes
DOD
44
10
M/65
T1N0M0
sublingual
S+ND+RT
pos.
yes
NED
80
11
M/45
T3N0M0
parotid
S+ND+RT
pos.
yes
NED
46
12
F/56
T3N0M0
parotid
S+RT
pos.
yes
AWD
172
13
F/79
T4N1M0
palat. tonsil
RT
N/A
no
DOD
7
14
F/77
T2N0M0
parotid
S+RT
neg.
yes
NED
22
16 17 18 19 20
M d
Ac ce pt e
15
cr
ip t
Sex/Age TNM staging
an
Pat.
us
Table 1. Clinicopathologic data of patients with SACC
F/49
T3N0M0
submand.
S+RT+CHT
neg.
yes
NED
162
F/76
T3N1M0
sublingual.
S+ND+RT
neg.
yes
NED
5
F/54
T1N0M0
submand.
S+RT
N/A
yes
DOD
130
M/51
T4N2bM0
max. alv. ridge
S+RT
neg.
yes
NED
86
M/24
T1N0M0
submand.
S+RT
neg.
yes
NED
282
M/61
T3N0M0
deep parotid
S+RT
pos.
yes
AWD
7
* Abbr. AWD - alive with disease, CHT - chemotherapy, CR - complete remission, DOD - dead of disease, F/U - follow-up, N/A - not available, ND - neck dissection, NED - no evidence of disease, RT - radiotherapy, S - surgery, SM - surgical margins
Page 19 of 31
20
Parameter*
Min
Max
Mean
SD
VEGF-C
0
5
3.65
1.25
VEGF-D
3
6
3.82
0.88
iLVD
1
15
6.32
3.83
pLVD
0
15
7.0
4.04
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Table 2. VEGF histoscore and LVD in SACC
* Abbr. iLVD - intratumoral lymphatic vessel density, pLVD - peritumoral lymphatic vessel density, SD - standard deviation,
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VEGF-C - vascular endothelial growth factor C, VEGF-D - vascular endothelial growth factor D
Page 20 of 31
21 Table 3. VEGF histoscores in acinar cells and LVD in normal parenchyma Parameter*
Min
Max
Mean
SD
VEGF-C
0
4
1.67
1.78
VEGF-D
0
5
2.33
1.92
LVD
0
8
1.75
2.56
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* Abbr. LVD - lymphatic vessel density, SD - standard deviation, VEGF-C - vascular endothelial growth factor C, VEGF-D -
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d
M
an
us
cr
vascular endothelial growth factor D
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Figure1a
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Figure1b
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Figure1c
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Figure2
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Figure3a
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Figure3b
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Figure4a
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Figure5
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