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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 3  |  Page : 521-526
Prognostic importance of microvessel density, VEGF expression and perineural invasion in laryngeal cancer treated with adjuvant radiotherapy


1 Department of Radiation Oncology, Basaksehir Cam and Sakura City Hospital, Istanbul, Turkey
2 Department of Radiation Oncology, Trakya University Medical Faculty, Edirne, Turkey
3 Department of Pathology, Trakya University Medical Faculty, Edirne, Turkey
4 Department of Radiation Oncology, Istinye University Medical Faculty, Arel University Medical Faculty, Istanbul, Turkey
5 Department of Radiation Oncology, Arel University Medical Faculty, Istanbul, Turkey

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Date of Submission25-Jun-2021
Date of Decision21-Jan-2022
Date of Acceptance30-Jan-2022
Date of Web Publication21-Jul-2022
 

   Abstract 


Background and Aims: We aimed to investigate the prognostic importance of the microvessel density (MVD) value, the vascular endothelial growth factor (VEGF) expression, and the presence of perineural invasion (PNI) in laryngeal cancer (LSCC) patients. Methods: Pathological specimens of 62 LSCC patients were assessed for the evaluation of the MVD value, the VEGF expression level, and the presence of PNI of the tumors. The tumor characteristics and prognostic effects of these parameters on local control (LC) and overall survival (OS) were analyzed. Statistical Analysis: Descriptive analyses were done using frequencies for the demographic variables. The survival estimates were calculated by the Kaplan–Meier survival curves. The effects of the parameters on LC and OS were investigated by using the log-rank test comparing the survival rates. Cox regression analysis was used for multivariable analysis. Results: The 5-year LC and OS rates of the 62 LSCC patients were 64.5 and 53.9%, respectively. Twenty-two patients (35.5%) had PNI and the frequency of PNI was higher in the patients with a high-grade disease (P = 0.01). The MVD value was higher in the tumors of older patients (P = 0.035) and was correlated with the VEGF expression (P = 0.009). A higher tumor grade was related to a higher VEGF expression (P = 0.01) and the increase in the VEGF expression was associated with a significant decrease in the OS (P = 0.03). Conclusion: The VEGF expression, the MVD value, and the presence of PNI had no prognostic significance on the LC in the LSCC patients while only the VEGF expression was associated with the OS.

Keywords: Microvessel density, perineural invasion, vascular endothelial growth factor expression

How to cite this article:
Harmankaya I, Çaloğlu VY, Taştekin E, Türkkan G, Çaloğlu M, Uzal C. Prognostic importance of microvessel density, VEGF expression and perineural invasion in laryngeal cancer treated with adjuvant radiotherapy. Indian J Pathol Microbiol 2022;65:521-6

How to cite this URL:
Harmankaya I, Çaloğlu VY, Taştekin E, Türkkan G, Çaloğlu M, Uzal C. Prognostic importance of microvessel density, VEGF expression and perineural invasion in laryngeal cancer treated with adjuvant radiotherapy. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 14];65:521-6. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/521/351612





   Introduction Top


Laryngeal cancer (LSCC) is a common subtype of head and neck cancer. There are approximately 1,77,000 new LSCC cases and 94,000 deaths due to LSCC annually.[1] A majority of the laryngeal cancers are squamous cell carcinomas.[2] Surgery, radiotherapy (RT), chemotherapy (ChT), and a combination of these modalities are the main treatment choices for non-metastatic LSCC.

Despite improvements in the treatment options, the treatment efficiency is not satisfactory, especially for locally advanced laryngeal tumors. The 5-year overall survival (OS) rates for locally advanced LSCC vary from 46.4 to 54.9%, depending on different treatment combinations.[3] Besides, the treatment results differ from one patient to another. Various prognostic pathological factors were identified and investigated for LSCC to predict which patients will benefit the most from which treatment strategy. Creating a marker system may be helpful to determine the prognosis and best treatment strategy of the LSCC patients.

Angiogenesis is a crucial step in tumor growth and metastasis. In the head and neck squamous cell carcinomas (HNSCC), vascular endothelial growth factor (VEGF)-like angiogenesis factors are highly expressed and are associated with the patient's prognosis. Although monotherapy with antiangiogenesis agents has generally demonstrated low or modest activity, they can induce radiosensitivity and chemosensitivity in combination therapies. The antiangiogenesis agents can regulate the tumor microenvironment, however, individualization of the treatment may be essential with the identification and validation of predictive biomarkers for targeted therapy.[4]

The microvessel density (MVD) assessments are based on the expression levels of the vascular endothelium markers such as CD34 or CD105 and correspond to the intensity of neoangiogenesis. The variations in the MVD data in the literature suggest identification of the reasons behind these inconsistencies. The most critical concerns are related to (a) the disease heterogeneity depending on several distinct sites of HNSCC and tumor histologies; (b) the variations in the quality and specificity of the antibodies used for MVD assessments; (c) the different methods for the tissue selection and utilization; and (d) the assessment algorithms that are based on the experience of the individual investigators. The assessment with a uniform immunohistochemistry (IHC) methodology of specific antibodies for all endothelial cell antigens may be necessary to adequately reflect the true MVD values in HNSCC.[5]

Perineural invasion (PNI) is a common histologic finding of the neoplastic invasion of the nerves in the head and neck cancer and is associated with poor clinical outcomes. The mechanisms underlying its pathogenesis remain largely unknown. The use of radiation therapy for PNI can improve local control (LC) but increases the risk of toxicity according to the location of the adjacent critical structures. Currently, the specific therapies of PNI are lacking despite the widespread acknowledgment of the prognostic significance.[6]

In this study, the MVD value, VEGF expression level, and presence of PNI of the tumors were analyzed in the LSCC patients who were treated with surgery followed by postoperative RT and/or ChT. The primary objectives of this study were to determine the characteristics of laryngeal tumors which may affect the treatment response rates negatively or positively and to be able to propose the best treatment option for the LSCC patients by considering their tumor characteristics.


   Materials and Methods Top


Sixty-two LSCC patients, who were referred for adjuvant oncological treatment at the Trakya University between September 1999 and June 2011, were included in this retrospective study. The study population was aged between 18 and 65 years and all the patients had surgery followed by postoperative RT and/or ChT.

The total laryngectomy and partial laryngectomy surgical specimens of all the patients were assessed by a pathologist for the evaluation of the MVD value, the VEGF expression level, and the presence of PNI of the tumors. The effectiveness and prognostic importance of these parameters were analyzed for each patient.

The ethical committee approval was received from the Ethics Committee of Trakya University Medical Faculty with the protocol code of TUTF-GOKAEK 2012/65, the decision number of 06/21, and the study was conducted according to the Helsinki Declaration and good clinical practice.

Radiotherapy

For the sites of suspected subclinical spread 45 Gy and for the sites of high-risk spread (in 33–37 fractions, once a day, for 5 days a week, 1.8 Gy per fraction) 59.4–66.6 Gy was prescribed.

The patients were evaluated once a week during their RT treatments and were followed up in compliance with the international guidelines after completion of their adjuvant treatments. The treatment responses were evaluated 12 weeks after the end of RT, both radiologically and endoscopically. The OS was calculated by the date of death or last seen in the outpatient clinic.

Pathological evaluation

The pathological specimens of the patients who were stained with hematoxylin-eosin were re-evaluated by a pathologist. Appropriate pathology blocks were chosen and immunohistochemical staining for the CD34 and VEGF antibodies was performed as described in the literature.[7],[8] The grading system recommended by the WHO was used to evaluate the differentiation of the squamous cell carcinoma (SCC). In our study, the WHO Classification of Head and Neck Tumors for the histological and genetic typing of human tumors, in the ninth volume of the fourth edition of the 2017 WHO series was used.

Tissue slides were incubated at 56°C for 2 h. Xylene series was applied three times for 10 min at 60°C and followed by 96, 80, and 70% alcohol and three times distilled water series, respectively. Citrate buffer 10X pH 8,0 (Kod: 15-M820, Lot. 50930) was used for antigen retrieval (heat in 95–100°C: 20 min/cool in room temperature: 20 min). PBS was applied for 10 min after endogen peroxide blocking with %3 H2O2. The slides were incubated with VEGF at room temperature for 32 min and CD34 antibodies for 56 min. Later, the immunoperoxidase staining method steps were applied.

In the immunohistochemical studies, kidney tissue was used as external positive control and intra-tissue vessels were used as internal positive control for both antibodies.

  1. Evaluation of the VEGF expression:


  2. All pathological sections were assessed by the pathologist. The positive cells were counted and examined. After this evaluation, the VEGF expression levels of the tumors were scored from 1 to 4 according to the staining intensity and the percentage of the positive tumor cells for immunohistochemistry. Each patient's VEGF expression result was classified into four levels as follows:

    Level 1: No positive cells means no expression (-)

    Level 2: <10% positive cells means slight staining/low expression (+)

    Level 3: 10–50% positive cells means medium staining/middle expression (++)

    Level 4: >50% positive cells means strong staining/high expression (+++)

  3. Evaluation of the MVD value:


  4. We used the Nikon Eclips 600 microscope for our evaluations. At first, the tumor area with the highest number of microvessels (hot spot) was detected under low magnification (X40–100 magnification). Second, the number of microvessels expressing CD34 was counted under high magnification (X200 magnification), diameter 1.1 mm and square area 0.95 mm; and the MVD value was assessed.

    An anti-CD34 antibody was used to stain the blood vessels. Any brown-stained endothelial cell or endothelial cell cluster that was separated from the adjacent microvessels, tumor cells, and other connective tissue elements, was considered as microvessels.

  5. PNI:


  6. The histopathologic examinations of the pathologic specimens were done by a pathologist with the use of a light microscope. The presence of PNI was evaluated under high magnification (40X objective and 10X ocular magnification). The pathologist screened all the sections from the tumor to identify the PNI evaluated as present/absent.


Statistical analysis

Descriptive analyses were done using frequencies for the demographic variables. The survival estimates were calculated by the Kaplan–Meier survival curves. The OS was calculated as the length of time from the date of diagnosis and LC was defined as the total disappearance of the primary tumor. The effects of the parameters on LC and OS were investigated by using the log-rank test comparing the survival rates. Cox regression analysis was used for multivariable analysis. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 17.0 (SPSS Inc., Chicago, IL, USA). A P value of less than 0.05 was considered statistically significant.


   Results Top


Among the 62 patients, 58 (93.5%) were males and the median age on diagnosis was 56 years (range: 36–65). The mean follow-up was 32.4 months (range: 2–125). On follow-up, 21 patients (33.8%) had local recurrence. At the end of the follow-up, 24 patients (38.7%) were still alive. The patient and disease characteristics are summarized in [Table 1].
Table 1: Patient and disease characteristics.

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Twenty (32.3%) patients had a history of alcohol consumption and a majority of the patients (91.9%) had a history of smoking. Most of the patients (67.7%) had T4 disease and the T-stage was significantly related to the tumor grade (P = 0.049). Twenty-four patients (38.7%) had N0, 18 patients (29%) had N1, and 20 patients (32.3%) had N2 disease, while none of the patients had N3 disease. The stage distribution of the patients was as follows: Stage II, 4 (6.5%); Stage III, 16 (25.8%), and Stage IV, 42 (67.7%) patients.

All of the patients had conventional SCC, including well-differentiated (Grade 1) in 16 (25.8%) patients, moderately differentiated (Grade 2) in 23 (37.1%) patients, and poorly differentiated (Grade 3) in 23 patients (37.1%). The 5-year LC and OS rates were 64.5 and 53.9%, respectively. [Figure 1] and [Figure 2] show the 5-year LC and OS rates.
Figure 1: The 5-year local control

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Figure 2: The 5-year overall survival

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On pathological examination, 22 patients (35.5%) had PNI [Figure 3]. The presence of PNI was not correlated with the tumor stage (both T and N stages), the VEGF expression, or the MVD value. The frequency of PNI was higher in the patients with a high-grade disease (P = 0.01). Ten (45%) of the 22 PNI-positive patients and 11 (27.5%) of the 40 PNI-negative patients had local recurrence at the end of the follow-up. The 5-year LC rates were 59% for PNI-negative and 43.9% for PNI-positive patients, respectively, and there was no significant difference in the 5-year LC between the PNI-negative and PNI-positive patients. The median survivals were 60 months for PNI-negative patients and 54 months for PNI-positive patients. At the end of the 5-year follow-up, 57.4% of the PNI-negative patients and 34.4% of the PNI-positive patients were alive, and there was no statistically significant difference in the 5-year OS between the two groups (P > 0.05).
Figure 3: Perineural invasion in the specimen of laryngeal cancer (LSCC) (X100)

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The mean MVD value was 33 (range: 12–55) [Figure 4]. This value was not associated with the tumor grade, tumor stage (both T and N stages), or the presence of PNI. The MVD value was higher in the tumors of the older patients (P = 0.035). Additionally, the MVD value was related to the VEGF expression (P = 0.009). Eight (33.3%) of the 24 patients with MVD value ≥33 and 13 (34.2%) of the 38 patients with MVD value <33 had local recurrence at the end of the follow-up. The 5-year LC rates were 62.9 and 47.7% for the patients with MVD value ≥33 and for patients with MVD value <33, respectively, and there was no significant difference in the 5-year LC between the two groups (P > 0.05). The 5-year OS rates were 48.9% for patients with MVD value ≥33 and 42.4% for the patients with MVD value <33, respectively, and there was no significant difference in the 5-year OS between the two groups.

The distribution of the VEGF expression levels of the patients was as follows: level 1, 4 (6.5%); level II, 7 (11.3%); level III, 24 (38.7%); and level IV, 27 (43.5%) patients [Figure 5]. The VEGF expression was not correlated with the tumor stage (both T and N stages) or the presence of PNI. A higher tumor grade was found to be associated with a higher VEGF expression (P = 0.01). [Table 2] shows this relationship. The 5-year LC rates were 50, 62.5, 54.4, and 54% for the patients with levels 1, 2, 3, and 4 VEGF expressions, respectively. No statistically significant relationship was found between the 5-year LC and the patient's VEGF expression levels. Furthermore, the 5-year OS rates were 75, 62.5, 43.2, and 38.9% for the patients with levels 1, 2, 3, and 4 VEGF expressions, respectively. The increase in the VEGF expressions was found to be associated with a significant decrease in the OS (P = 0.03). Therefore, the tumor stage, the presence of PNI, and the 5-year LC were not found to be correlated with the VEGF expression in multivariate analysis. However, the tumor grade and 5-year OS were detected in relation with the level of VEGF expression.
Figure 4: Representative immunohistochemical staining of CD34; (a) low microvessel density (MVD) (X100) (b) high MVD (X50)

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Figure 5: Representative immunohistochemical staining of the vascular endothelial growth factor (VEGF) in the tumor cells; (a) poor staining (<10% positive cells) (X200), (b) moderate staining (10–50% positive cells) (X100), (c) strong staining (>50% positive cells) (X100)

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Table 2: The relationship between tumor differentiation grade and the VEGF expression level.

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   Discussion Top


Despite the intense and combined treatments for locally advanced LSCC, the 5-year OS rates are still not satisfactory. Several prognostic pathological factors have been analyzed to predict which patients will benefit the most from which treatment strategy. We assessed the pathological prognostic factors such as PNI, VEGF, and MVD. There are some controversies in the literature about the roles of PNI, VEGF, and MVD in the prognosis of LSCC.

The prognostic importance of PNI in LSCC has been evaluated in many studies. The perineural space invasion is a negative prognostic factor associated with high rates of locoregional recurrence and low disease-free survival rates. A surgery-alone approach is usually not sufficient to treat laryngeal cancer with PNI. Minor laryngeal nerve involvement is expected more than superior and inferior laryngeal nerve involvement. It showed that laryngeal cancer patients with PNI deserve a closer clinical and instrumental follow-up. In the treatment of these cases, it is important to use adjuvant radiotherapy (RT) following surgical treatment.[6] In their multi-ethnic primary HNSCC cohort study, Sethi et al.[9] examined the histopathology and demographic variables at primary diagnosis in 428 patients—171 of which were laryngeal cancers. PNI was detected in 30.2% of the patients and PNI was one of the independent risk factors for advanced-stage disease in multivariable analysis. Similarly, PNI was detected in 35.5% of our patients. On the other hand, we did not observe a relationship between PNI and advanced-stage disease but the frequency of PNI was higher in the patients with a high-grade disease. The relatively small number of patients in our study may explain this difference. Additionally, PNI has been reported as a poor prognostic factor in the studies of LSCC regarding LC and OS.[10],[11] In contrast to these studies, there was no relationship between PNI and LC and OS in our study. This discrepancy may also be explained by the relatively small number of patients in our study.

Angiogenesis is an important factor for normal tissue development/repair as well as tumor development. As a response to hypoxia, angiogenesis becomes activated and can be rearranged, and many heterogeneous vessels may occur.[12] These courses have been evaluated in several cancer types by the measurement of VEGF and/or MVD. In their meta-analysis, Kyzas et al.[13] assessed the relationship between the VEGF expression and clinical outcomes in patients with HNSCC. The VEGF positivity was associated with worse OS and there was a trend toward a correlation of the VEGF positivity with a higher stage and poor differentiation. Consistent with this study, we found a significant correlation between higher VEGF expression levels and worse OS. Additionally, a higher VEGF expression was associated with higher tumor grade, but not with the tumor stage.[14] The blockade of the VEGF receptor as a method of targeted therapy is supported by this evidence.[15] In another study, Parikh et al.[14] reported that the VEGF expression plays an uncertain role in LC but may be a predictive factor for the overall prognosis. Similar to this study, we also did not find a significant correlation between the VEGF expression and LC, but the VEGF expression was associated with a significant decrease in the OS.

The demographic features and clinicopathologic importance of MVD have been assessed in several tumor types. In general, MVD was shown to be associated with metastasis and prognosis and was mostly predictive in the breast, prostate, and hematological malignancies.[16] Additionally, the mean value of MVD was reported to be higher in older patients.[17] Similar to this study, we also observed higher MVD values in older patients in our study. Furthermore, the MVD value was associated with the VEGF expression in our study and this result was in concordance with some previous studies.[18],[19]

While considering the density of tumor angiogenesis as a prognostic factor, the potential correlations of microvessel density with primary lesion stage (T stage), the process of metastasis, and locoregional recurrences should be questioned. In the literature of this widely studied area in oncology, there are controversial findings for these correlations.[20]

Even though there are studies demonstrating the significant association between MVD and LC and OS, there are also some incompatible results about the role of MVD in the prognosis of laryngeal cancers. The literature provides controversial information about this subject. Some studies showed a significant relationship between the higher microvessel count and local relapse and the presence of metastasis.[21],[22] Furthermore, a significant correlation between MVD and tumor stage (both T and N stages), tumor grade, and OS was reported.[23] In contrast with these studies, some studies revealed no correlation between MVD and LC and OS in the LSCC patients.[24],[25],[26] In our study, no correlation was found between MVD and LC and OS, but this result may change if we had a larger study population.

Nevertheless, our study has some limitations as well. This is a retrospective study and our patient number is relatively small. Additionally, patients were treated with older RT devices and techniques which probably have more uncertainty in terms of imaging and planning techniques in comparison with modern devices and techniques. Currently, the LC and OS results are expected to be slightly better with the use of sophisticated imaging and planning techniques in radiotherapy.


   Conclusions Top


In conclusion, this retrospective study showed that the VEGF expression, the MVD value, and the presence of PNI had no prognostic significance on the LC in the LSCC patients who were treated with primary surgery followed by postoperative RT and/or ChT. Among these three pathological parameters, only the VEGF expression was associated with a decrease in the OS. Even though the literature provides controversial information, large randomized prospective studies using common pathological evaluation methods may elucidate the prognostic effects of these three pathological parameters in the LSCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Ilknur Harmankaya
Department of Radiation Oncology, Basaksehir Cam and Sakura City Hospital, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_655_21

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    Figures

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