Introduction:
Lymphovascular invasion (LVI), which refers to the invasion of lymphatic spaces, blood vessels or both in the peritumoral area by tumour emboli, is one of the critical steps in metastasis. The prognostic value of LVI in breast cancer was first described more than four decades ago (1). Subsequently, several independent studies have shown a relationship between LVI and outcome in predominantly patients with negative, and much lesser association in positive, lymph node (LN) status (2,3). Routine assessment of LVI is now part of the minimum data set for breast cancer pathology reporting produced by the United Kingdom Royal College of Pathologist, the European Commission, and the College of American Pathologists. It is endorsed by the World Health Organization, the St. Galen Consensus Conference and the American Joint Committee on Cancer/International Union against Cancer (AJCC/UICC) (seventh edition of the TNM classification) as a prognostic factor in patients with breast cancer (2,4). However, LVI is not incorporated in most of internationally recognized staging systems, such as the AJCC/UICC TNM staging system (2,4,5), the prognostic algorithms and indices such as the Adjuvant Online, Predict, and the Nottingham Prognostic Index [NPI] and the adjuvant chemotherapy guidelines such as the North Central Cancer Network breast cancer treatment guidelines and the American Society of Clinical Oncology breast guidelines.
The utility of LVI in clinical management decisions remains a matter of debate for the following reasons: 1) most studies of LVI have been small or only used univariate analysis (2,4, 6); 2) variation with regard to distinguishing lymphatic invasion from blood vessel invasion or consideration of the extent of LVI (7-9); 3) the use of different methods for the detection of LVI, including pathologic routine assessment and immunohistochemistry (10,11) and 4) few studies that included large series of breast cancer either lacked details of the significance of LVI in the different clinically relevant subgroups, did not report its risk value compared with other well established prognostic markers, or reported a lack of significance (12).
In the present study, we aimed to determine the association and correlation between lymphovascular invasion with clinical staging and histological grading in retrospective cohort samples. We also aimed to determine whether the presence or absence of LVI contributed to any significant information in predicting survival of patients with invasive breast cancer.
Materials and Methods
This is a retrospective cohort study using data obtained from reviewing archival histological material and patient’s medical record. All female patients aged 85 years old and/or less diagnosed with invasive breast carcinoma at all stages from January 2005 to December 2013 were included in this study. This study was approved by the UKM Institutional Review Ethics Committee/IRB (UKM 1.5.3.5/244/FF-138-2013).
The cases were obtained from the data of National Cancer Registry 2005-2013 from the records available at Histopathology Unit and Surgical Unit, Queen Elizabeth Hospital (QEH), Sabah, Malaysia. The information regarding patient’s age, ethnic, staging of the tumor at diagnosis were recorded. Tumour stages were categorized into two main stages: low stage (stages I and II) and high stage (stages III and IV). Patient’s clinical outcome five years after first diagnoses was also reviewed which includes breast cancer specific survival (BCSS) (calculated as the time from the date of surgery to the date of death or alive at last follow-up) or distant metastasis free survival (DMFS) (calculated as the time from the date of surgery to the date of disease recurrence). Both BCSS and DMFS were obtained from the patient’s file at Surgical Unit and medical record office, QEH.
Tumour grading, LN status, hormonal [oestrogen (ER), progesterone (PR) and HER2] status lymphovascular invasion, were obtained from Breast Cancer Proforma and histopathology report at Histopathology Unit, QEH. Tumour grading was categorized into low (grades 1 and 2) and high (grade 3) grade. Patient’s data was made anonymous and each subject was coded accordingly.
Assessment of lymphovascular invasion
All breast cancer cases with or without reported LVI were selected. In this study, LVI was assessed in the peritumoral tissue on haematoxylin and eosin (H&E) stained sections. LVI was defined as carcinoma cells present within an endothelial-lined space (lymphatic). Some of the cases without reported LVI show possible LVI (tumor cell emboli observed in a space with the appearance of a vessel but without a recognizable endothelial lining) and this was scored as negative. Further immunohistochemistry of D2-40 staining, a lymphatic vessel marker, was done on the LVI negative cases to confirm and/or exclude LVI. The presence of intraluminal tumor cluster completely surrounded by strong cytoplasmic D2-40 expression by lymphatic endothelial cells was considered as positive for lymph vessel invasion. The results were analyzed as lymph vessel invasion negative or positive. Tissue from normal/noncancerous area of the same mastectomy or wide excision biopsy specimen was used as control for D2-40 immunohistochemistry.
D2-40 Immunohistochemistry
D2-40 is a monoclonal mouse anti-D2-40 which identifies a 40kD O-linked sialoglycoprotein present on a variety of tissues including fetal testes and testicular germ cell tumors. D2-40 has also been demonstrated to react with lymphatic endothelium and is unreactive with vascular endothelium. In neoplastic tissue, immunostaining of lymphatic endothelium by D2-40 has been shown to be useful in identifying lymphatic invasion of primary tumor. Preparation of tissue and slides, immunohistochemical procedure, preparation of controls and determination of the positivity and negativity of the staining were according to the standards and guidelines recommended by manufacturing company.
Formalin fixed paraffin embedded blocks of the chosen invasive breast carcinoma with no lymphovascular invasion were taken from the histopathological archive of the Department of Pathology, Queen Elizabeth Hospital. The sections were incubated with a primary antibody D2-40 (Dako, clone D2-40). The primary antibodies were diluted to a final dilution of 1:1200 and 1:200, respectively. Dako antibody diluent (S0809) was used. The EnVisionFlex detection system (Dako, K8000) was used for visualization. Sections were incubated for 5 min with peroxydase-blocking reagent (SM801), 20 min with the EnVision FLEX/HRP Detection Reagent (SM802), 10 min with EnVision FLEX DABþ Chromogen (DM827) / EnVision FLEX Substrate Buffer (SM803) mix and 5 min with EnVision FLEX Hematoxylin (K8008). The slides were then dehydrated and mounted. All immunohistochemical stainings were performed using a Dako Autostainer Link 48 instrument and EnVision FLEX Wash Buffer (DM831).
Statistical analysis
A computer program using SPSS program version 21.0 was used to process and analyze the data. The data was non-parametrical of nominal and ordinal (categorical) type. The result was analyzed statistically by using chi-square test. Spearman correlation test was done in data with significant association. In survival analysis, Kaplan-Meier estimates and comparison using log rank tests was done to compare survival curves between groups. Cox-regression test was done to compare with covariate information. The statistical significance was set at p <0.05.
Results
LVI was detected in 87 out of 117 (74.4%) cases and negative in 30 (25.6%) cases. Of these cases, 71 (60.7%) were of stage pT1-2 and 46 (39.3%) were of stage pT3-4. A total of 85 (72.6%) cases had more than three lymph nodes involvement (stage pN2-3) while 32 (27.4%) cases showed less than three lymph nodes involvement (stage pN0-1). There was evidence of distant metastasis in 24 cases (20.5%) while 93 (79.5%) cases lacked metastasis. Histological grading showed that 68 (58.1%) cases were of low grade and 49 (41.9%) cases were of high grade. D2-40 staining on cases with LVI negative on H&E showed 10 out of 30 (33.3%) cases positive for LVI while 20 out of 30 (66.7%) cases were negative for D2-40, confirming absence of LVI.
A total of 95 (81.2%) cases were available for the BCSS analysis. From this group of population, 85 (89.5%) cases were alive during their last follow up while 10 (10.5%) cases were dead five years or less after their first diagnosis. A total of 22 (18.8%) cases were available for DMFS analysis, of which 19 (86.4%) cases had distant metastasis and 3 (13.6%) had tumor recurrence.
Association-correlation between LVI and staging and grading in invasive breast carcinoma
The association between LVI and staging and histological grading is shown in Table 1. LVI was significantly associated with lymph node involvement and distant metastasis (P value <0.05). LVI showed no significant association with tumour size (cm) and histological grading (P value >0.05).
| Table 1. Association between lymphovascular invasion and staging and grading. |
Variable |
No. of patients (%) |
Chi-square statistics |
*p value |
| |
LVI negative |
LVI positive |
|
|
Total |
30 (25.6%) |
87 (74.4%) |
|
|
Tumour size |
pT1-2(<5 cm) |
51 (71.8%) |
20 (28.2%) |
.605 |
.437 |
pT3-4 (>5cm) |
36 (78.3%) |
10 (21.7%) |
Lymph node involvement |
pN0-1 (≤3) |
13 (40.6%) |
19 (59.4%) |
26.290 |
.000 |
pN2-3 (>3) |
74 (87.1%) |
11 (12.9%) |
Distant metastasis |
M0 |
65 (69.9%) |
28 (30.1%) |
4.744 |
.029 |
M1 |
22 (91.7%) |
2 (8.3%) |
Clinical stage |
Low stage |
56 (70.0%) |
24 (30.0%) |
2.521 |
.112 |
High stage |
31 (83.8%) |
6 (16.2%) |
Histological grade |
Low grade |
48 (70.6%) |
20 (29.4%) |
1.211 |
.271 |
High grade |
39 (79.6%) |
10 (20.4%) |
| *p<0.05 was considered significant |
Survival analysis
In BCSS, the probability of survival was better in cases with positive LVI in the first 40 months. However, survival became poor after 40 months of study interval. In DMFS, the probability of survival was poor in cases with negative LVI. However, LVI was not a significant factor in both subgroups of patients (P>0.05).
However, further survival analysis showed that LVI was a significant prognostic factor in two subgroups of BCSS - alive at last follow up and DMFS-metastasis (P<0.05). The result is illustrated in Figure 1.
 |
 |
Figure 1: Survival curve chart for subgroups of a) BCSS-last follow up and b) DMFS-metastasis with lymphovascular invasion as a prognostic factor. |
Multivariate cox regression analysis of factors associated with breast cancer-specific (BCSS) and distant metastasis-free survival (DMFS)
Multivariate cox regression analysis showed LVI (HR, 1.5; 95% CI, 1.0-2.5; P = .046) was a significant prognostic factor associated with BCSS but not a significant prognostic factor associated with DMFS (HR, 3.5; 95% CI, 0.8-15.3). Distant metastasis (HR, 29.7; 95% CI, 9.9-89.2; P = .000) was a significant prognostic factor associated with DMFS. These findings are shown in Table 2 and illustrated in Figures 2 and 3. Additionally, the analysis also showed poorer outcome after 5 years follow up in cases with positive LVI and presence of distant metastasis (M1).
| Table 2. Association of multiple prognostic factors with BCSS and DMFS |
Prognostic factor |
BCSS |
DMFS |
| |
HR (95% CI) |
p |
HR (95% CI) |
p |
Lymphovascular invasion |
1.5 (1.0-2.5) |
.046 |
3.5 (0.8-15.3) |
.085 |
PR status |
1.5 (1.0-2.4) |
.034 |
2.2 (0.9-5.3) |
.061 |
Distant metastasis |
1.4 (0.6-3.2) |
.417 |
29.7 (9.9-89.2) |
.000 |
| *p<0.05 was considered significant |
 |
 |
Figure 2: Survival curve chart for subgroups of BCSS with a) LVI and b) PR status. |
 |
Figure 3: Survival curve chart for subgroups of DMFS with metastasis. |
This analysis also showed clinical staging (HR, 13.6; 95% CI, 2.8-64.6; P = 0.001), tumour subtype (HR, 12.1; 95% CI, 2.0-73.4; P = 0.007) and histological grading (HR, 5.8; 95% CI, 1.2-26.6; P = 0.023) were significant prognostic factors associated with BCSS-Death. However, only age (HR, 1.7; 95% CI, 1.1-2.6; P = 0.014) was as a significant prognostic factor associated with BCSS-Last Follow Up (Table 3, Figures 4 and 5). Poor outcome following 5 years of follow-up was associated with higher clinical stage, tumour subtype of invasive ductal carcinoma (IDC) and higher histological grade.
| Table 3. Significant prognostic factors associated with Breast Cancer Specific Survival (BCSS) subgroups. |
Predictor |
Death |
Last Follow up |
| |
HR (95% CI) |
p |
HR (95% CI) |
p |
Age |
1.7 (0.4-6.8) |
.412 |
1.7 (1.1-2.6) |
.014 |
Clinical staging |
13.6 (2.8-64.6) |
.001 |
1.8 (0.8-4.0) |
.122 |
Specimen subtype |
12.1 (2.0-73.4) |
.007 |
1.5 (0.9-2.4) |
.081 |
Histological grading |
5.8 (1.2-26.6) |
.023 |
1.3 (0.8-2.2) |
.151 |
| *p<0.05 was considered significant |
Specimen type (HR, 9.9; 95% CI, 1.3-75.0; P= 0.025), tumor size (cm) (HR, 7.5; 95% CI, 2.5-22.7; P=0.000 and distant metastasis (HR, 10.6; 95% CI, 1.4-81.1; P = 0.022) were also found to be a significant prognostic factor associated with DMFS. On the other hand, tumour recurrence was not found to be a significant prognostic factor associated with DMFS. Patients with mastectomy, larger tumor size (pT3-4) and distant metastasis (M1) also showed poorer outcome after 5 years of follow up (see Table 4, Figure 6).
| Table 4. Prognostic factors associated with DMFS subgroups. |
Predictor |
Recurrence |
Metastasis |
| |
HR (95% CI) |
p |
HR (95% CI) |
p |
Specimen type |
0.02 (0.0-1030.4) |
.507 |
9.9 (1.3-75.0) |
.025 |
Tumor size (cm) |
0.008 (0.0-166.4) |
.339 |
7.5 (2.5-22.7) |
.000 |
Distant metastasis |
28 (0.0-6440279.2) |
.597 |
10.6 (1.4-81.1) |
.022 |
| *p<0.05 was considered significant |
 |
 |
 |
Figure 6: Survival curve chart showing association between DMFS and a) specimen type, b) tumor size(cm) and c) distant metastasis. |
Discussion
This study demonstrated that LVI had significant association and correlation with lymph node involvement and distant metastasis. However, LVI was not significantly associated with tumour size (cm) and histological grade. In this study, LVI was a significant prognostic factor in two subgroups of BCSS-alive at last follow-up and DMFS-metastasis for 5 years follow-up. In both subgroups, patients with LVI negative were more likely to survive compare to those with LVI positive. This finding was also consistent with that of a previous study where vascular invasion alone maintained its association with poorer BCSS and DMFS at 5- year follow-up (4).
The multivariate analysis also showed PR status were significant prognostic factor (P<0.05) in BCSS, where patients with PR positive were more likely to survive compare to those with PR negative. The presence of distant metastasis (M1) showed significant prognostic factor (P<0.05) in DMFS, where patients with no metastasis were more likely to survive compare to those with metastasis.
This analysis was further done in subgroups of both BCSS and DMFS. The clinical staging, tumour subtype and histological grading were significant prognostic factors associated with BCSS-Death. Higher clinical stage, tumour subtype of invasive ductal carcinoma (IDC) and higher histological grade were less likely to survive in 5- years follow up, suggesting these prognostic factors contribute to the higher risk of death, in keeping with those of previous findings(4). Age is also a significant prognostic factor associated with BCSS-Last Follow Up suggesting patients greater than 50 years old do not tend to come for follow up after 5 years of diagnosis and treatment.
Specimen type, tumour size (cm) and distant metastasis were significant prognostic factors associated with DMFS-Metastasis. It was previously reported that patients with mastectomy, larger tumor size (pT3-4) and presence of distant metastasis (M1) were less likely to survive after 5- years of follow-up, (4). However, we could not find any significant prognostic factor associated with DMFS-Recurrence.
Lymph node negative (pN0), tumour size (cm), ER status, HER2 status and the use of immunohistochemistry for detecting an endothelial-specific marker contributed to the prognostic significance of lymphovascular invasion (4). However, these variables were not significant prognostic factors in our study for both BCSS and DMFS groups and subgroups, possibly due to heterogeneity of study population present in this study.
In conclusion, LVI status has proven to be a reliable prognostic marker and thus incorporation of its status in the breast cancer staging system is strongly advocated. The present study demonstrated significant correlation between LVI and lymph node involvement with distant metastasis. LVI and PR status have independent prognostic significance particularly in the BCSS. The clinical staging, tumour subtype and histological grading were shown to be independent predictors of death. Distant metastasis has independent prognostic significance particularly in the DMFS and also in the metastasis subgroup. There was no significant correlation between LVI and histological grading. The use of D2-40 immunohistochemistry as a marker for LVI did not prove to be significant in predicting prognosis in patients with breast cancer. From the findings of the present study, we strongly advocate the incorporation of LVI status into the breast cancer staging system.
Conclusion
LVI correlates with clinical staging and is a reliable predictor of outcome in patients with invasive breast carcinoma and thus should be incorporated into breast carcinoma staging system.
Acknowledgement
This research was funded by the Fundamental Research Grant UKM Medical Centre (Grant code PPUKM, FF-138-2013) and approved by the Ethics Committee/IRB of UKM Medical Centre (UKM 1.5.3.5/244/FF-138-2013) and the Ministry of Health Malaysia. The authors declare that they have no competing interests.
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