Background: Lymphovascular invasion (VI) is an established prognostic marker for many cancers including bladder cancer. There is a paucity of data regarding whether the prognostic significance of lymphatic invasion (LVI) differs from blood vessel invasion (BVI). The aim was to examine LVI and BVI separately using immunohistochemistry (IHC), and investigate their associations with clinicopathological characteristics and prognosis. A secondary aim was to compare the use of IHC with assessing VI on standard HAS (hematoxylin-azophloxine-saffron) sections without IHC. Methods: A retrospective, population –based series of 292 invasive bladder cancers treated with radical cystectomy (RC) with curative intent at Vestfold Hospital Trust, Norway were reviewed. Traditional histopathological markers and VI based on HAS sections were recorded. Dual staining using D2–40/CD31 antibodies was performed on one selected tumor block for each case. Results: The frequency of LVI and BVI was 32 and 28%, respectively. BVI was associated with features such as higher pathological stages, positive regional lymph nodes, bladder neck involvement and metastatic disease whereas LVI showed weaker or no associations. Both BVI and LVI independently predicted regional lymph node metastases, LVI being the slightly stronger factor. BVI, not LVI predicted higher pathological stages. BVI showed reduced recurrence free (RFS) and disease specific (DSS) survival in uni-and multivariable analyses, whereas LVI did not. On HAS sections, VI was found in 31% of the cases. By IHC, 51% were positive, corresponding to a 64% increased sensitivity in detecting VI. VI assessed without IHC was significantly associated with RFS and DSS in univariable but not multivariable analysis. Conclusions: Our findings indicate that BVI is strongly associated with more aggressive tumor features. BVI was an independent prognostic factor in contrast to LVI. Furthermore, IHC increases VI sensitivity compared to HAS.
Keywords: Bladder cancer; Blood and lymph vessel invasion; CD31; D2–40
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1186/s13000-021-01171-7.
In bladder cancer (BC), the detection of vascular invasion (VI) by tumor tissue in RC specimens has been shown to be associated with adverse outcomes [[
The aim of this study was to examine the prevalence and prognostic significance of BVI and LVI assessed by IHC, and their associations with respect to subsequent metastatic disease and survival, in a population- based series of invasive bladder tumors (stage pT1–4) in RC specimens.
We studied a consecutive, population-based series treated with RC at Vestfold Hospital Trust. Since 2013, the hospital has had a multi-regional center function for cystectomy in patients with BC, covering regions in South-Eastern Norway comprising approximately 20% of the Norwegian population with around 1,060,000 residents.
This series accounted for 292 patients with invasive BC diagnosed from transurethral resection for bladder tumor (TURBT) treated with RC with curative intent between 2000 and 2018. Patients with distant metastases at the time of diagnosis, no residual malignant tissue in the cystectomy specimen (ypT0), concomitant ureter cancer or urachal adenocarcinomas were excluded.
Regarding primary treatment, 48 patients (16%) received neoadjuvant chemotherapy and 24 patients (8%) received adjuvant chemotherapy. We did not have any records on palliative radiation or chemotherapy during follow up.
The median follow up period was 48 months (range 19–180). None of the patients was lost to follow up due to insufficient information.
The clinical and follow up data were collected from the Urological Register at Vestfold Hospital Trust, and medical records at Vestfold Hospital Trust and referring hospitals with respect to disease recurrences, survival time, date for last status recorded (date, month and year) and deaths.
The cause of death was recorded based on evaluation of medical certificates of death obtained from medical journals, and in a few cases on telephone calls to general practitioners and nursing home doctors. In a very few cases autopsy had been performed. Occurrence of any relapse and/or metastatic spread during the course of disease was recorded with date and site based on evaluation of CT scans reported in the medical journals. Only in a very few cases the metastases were histologically verified by biopsy. For 44 patients the follow up information was obtained from a questionnaire sent by postal service.
All original histological sections were reviewed by one pathologist (BC). Traditional prognostic histopathological parameters such as tumor type according to WHO 1973/ISUP 2004 [[
As for divergent differentiation within the urothelial carcinoma category, the cases were divided into four groups. The groups were subsequently fused into two; micropapillary and other variants. We did not record the percentage of divergent histology within the tumors. The presence of VI based on HAS sections was evaluated according to the morphological criteria defined by Algaba [[
Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. All original sections were reviewed, and one representative block from the bladder wall with tumor tissue including deepest invasive focus and adjacent peritumorous tissue, if any, was selected for further staining. In all cases, the selected block was equal to a block with observed VI.
Immunohistochemical staining was performed on Benchmark Ultra (Ventana, Roche) on standard 4–5 μm sections, after overnight baking at 60 °C. Dewaxing and antigen retrieval was preserved in the instrument (fully automated, "on board"). Dual staining with Podoplanin, clone D2–40 (M3619, Dako, Agilent) for lymphatic vessel staining and CD31 (clone JC70A, M0823, Dako, Agilent) for blood vessel staining was performed. Briefly, the sections were pretreated with ULTRA Cell conditioning 1 (ULTRA CC1) for 36 min at 95 °C and then incubated with D2–40, diluted 1:25, for 32 min at 37 °C. Staining for D2–40 was carried out using UltraView DAB (ref. no.760–500). As for CD31, the sections were incubated for 32 min at 37 °C, diluted 1:40. Staining was carried out using UltraView Red (ref. no. 760–501).
Finally, the sections were counterstained with hematoxylin for 1 min (Shandon Instant Hematoxylin Kit, ThermoScientific).
All stained sections were examined by a pathologist (BC), and the observer was blinded for clinicopathological information. A few cases difficult to interpret were consulted with another experienced pathologist (TAK), and consensus was reached. We recorded LVI to be present if tumor tissue, i.e. single tumor cells, clusters of tumor cells or tumor thrombi were located within one or more than one D2–40 positive structure with weak or negative CD31 staining. BVI was reported when single tumor cells, clusters of tumor cells or tumor thrombi were detected in one or more than one CD31-positive and D2–40 negative vessel (Fig. 1).
Graph: Fig. 1 Histological images of LVI and BVI by immunohistochemistry (× 400). Footnotes: A, VI, HAS section. B, the same vessel, positive for D2–40 (LVI). C, VI, HAS section. D, the same vessel, positive for CD31 (BVI). E, VI, HAS section. F, the same vessel, positive for CD31 (BVI)
2 × 25 cases were arbitrarily chosen and reviewed by two pathologists for assessing Kappa values (Cohen's kappa coefficient) for interobserver reliability. As for the assessment of VI based on HAS sections, the Kappa value was 0.6, whereas BVI and LVI based on IHC showed very good agreement, with Kappa values of 0.9 and 1.0, respectively.
All statistical analyses were performed by using IBM SPSS Statistics, V. 26,0 (IBM, Armonk, USA). 292 patients were available for analyses. Associations between different categorical variables were assessed by Pearson's χ
Study participants submitted broad, informed consent, and data were de-identified for the analyses. The study was approved by the Regional Ethics Committee of South Eastern Norway (REK 2019/532).
Table 1 presents the patient characteristics of the study cohort, in total and stratified by gender; 68 (23%) were female, 224 (77%) were male. The median age at cystectomy was 72 years. In 256 patients (88%), regional lymph node dissection (LND) had been performed, of which 86 (34%) had lymph node metastases. Urothelial carcinomas comprised 274 (94%) of cases. Positive surgical margins were observed in 29 specimens (10%).
Table 1 Patient characteristics for the study cohort, in total and stratified by gender
Total (n,%) Female (n,%) Male (n,%) 292 68 (23) 224 (77) 40–68 103 (35) 24 (36) 79 (35) 69–76 95 (33) 20 (29) 75 (34) 77–88 94 (32) 24 (35) 70 (31) 68 (23) 224 (77) pT1 39 (13) 6 (8.8) 33 (15) pT2a 46 (16) 9 (13) 37 (17) pT2b 27 (9,2) 5 (7.4) 22 (9.8) pT3a 63 (23) 13 (19) 50 (22) pT3b 83 (28) 25 (37) 58 (26) pT4a 33 (11) 10 (15) 23 (10) pT4b 1 (0.3) 0 1 (0.4) 256 (88) 57 (84) 199 (89) N0 170 (66) 41 (72) 129 (65) N1–3 86 (34) 16 (28) 70 (35) 17 (11–17-22) 16 (11–16-22) 17 (11–17-22) Positive surgical margins 29 (10) 6 (8.8) 23 (10) CIS in urethral and/or ureteric margins 8 (2.7) 3 (4.4) 5 (2.2) Urothelial carcinoma 274 (94) 61 (90) 213 (95) Squamous cell carcinoma 11 (3.8) 5 (7.4) 6 (2.7) Adenocarcinoma 2 (0.7) 1 (1.5) 1 (0.4) Small cell neuroendocrine carcinoma 5 (1.7) 1 (1.5) 4 (0.8) 113 (41) 31 (51) 82 (39) Squamous 61 (22) 17 (28) 44 (21) Micropapillary 23 (8.4) 4 6.6) 19 (8.9) Glandular 6 (2.2) 1 (1.6) 5 (2.3) Others/mixed 23 (8.4) 9 (15) 14 (6.6) 136 (47) 25 (37) 111 (50) Trigone ± ureteric orifices 32 (11) 9 (13) 23 (10) Bladder neck 49 (17) 7 (10) 42 (19) Diverticula 12 (4.1) 0 12 (5.4) Other 199 (68) 52 (77) 147 (66) Neoadjuvant 47 (16) 11 (16) 36 (16) Adjuvant 24 (8.2) 4 (5.9) 20 (8.9)
Out of 292 patients, we observed relapse of disease in 142 (49%), of which 56 events were local recurrences, 33 regional lymph node metastases, 42 non-regional lymph node metastases and 140 various distant metastases. Among the 166 patients (57%) who died during the follow up period, 132 (80%) died from bladder cancer and 34 (20%) died from other causes.
Based on HAS sections, VI was found in 31% (n = 91) of cases and was significantly associated with histopathological variables such as higher pathologic stages (OR 5.8, CI 3.0–11.1, p < 0.001), regional lymph node metastases (OR 5.2, CI 2.9–9.2, p < 0.001), micropapillary variant histology (OR 2.8, CI 1.2–6.8, p = 0.019), bladder neck involvement (OR 3.4, CI 1.8–6.5, p < 0.001), positive surgical margins (OR 3.6, CI 1.6–7.9, p = 0.001), local recurrence (OR 2.1, CI 1.2–3.7, p = 0.006) and distant metastases (OR 3.2, CI 1.9–5.5, p < 0.001) (Table 2). By IHC, 150 (51%) patients were positive. Regarding treatment, LVI was associated with patient subgroup treated with adjuvant chemotherapy (OR 2.4, CI 1.0–5.5, p = 0.042). As for BVI and VI assessed by HAS, no associations were found regarding neither neoadjuvant nor adjuvant treatment.
Table 2 Associations between different categories of vascular invasion with and without IHC and various clinicopathological variables
BVI LVI VI Variables Neg (n,%) Pos (n,%) OR (95% CI) Neg (n,%) Pos (n,%) OR (95% CI) Neg (n,%) Pos (n,%) OR (95% CI) 211 (72) 81 (28) 200 (69) 92 (32) 201 (69) 91 (31) 40–72 120 (57) 34 (42) 1 107 (54) 47 (51) 1 112 (56) 42 (46) 1 73–88 91 (43) 47 (58) 1.8 (1.1–3.1) 0.022 93 (46) 45 (49) 1.1 (0.7–1.8) 0.70 89 (44) 49 (54) 1.5 (0.8–2.4) 0.13 50 (24) 18 (22) 45 (23) 23 (25) 1 49 (24) 19 (21) 1 161 (76) 63 (78) 0.9 (0.5–1.7) 0.79 155 (77) 69 (75) 1.1 (0.6–2.0) 0.64 152 (76) 72 (79) 0.8 (0.5–1.5) 0.51 t1 + 2 101 (48) 11 (14) 1 83 (42) 29 (32) 1 99 (49) 13 (14) 1 t3 + 4 110 (52) 70 (86) 5.8 (2.9–11.7) < 0.001 117 (58) 63 (68) 1.5 (0.9–2.6) 0.10 102 (51) 78 (86) 5.8 (3.0–11.1) < 0.001 N0 140 (74) 30 (45) 1 130 (73) 40 (51) 138 (78) 32 (41) 1 N1–3 49 (26) 37 (55) 3.5 (1.9–6.3) < 0.001 48 (27) 38 (49) 2.6 (1.5–4.5) 0.001 39 (22) 47 (60) 5.2 (2.9–9.2) < 0.001 Other (ref. gr. UTC, no diverg diff) 123 (89) 38 (86) 1 115 (91) 46 (81) 1 116 (91) 45 (79) 1 Micropapillary 16 (7.4) 6 (14) 1.2 (0.4–3.3) 0.71 11 (9) 11 (19) 2.5 (1.0–6.2) 0.042 11 (8.7) 12 (21) 2.8 (1.2–6.8) 0.019 Other 189 (90) 54 (67) 1 171 (86) 72 (78) 1 179 (89) 64 (70) 1 Bladder neck 22 (10) 27 (33) 4.3 (2.3–8.1) < 0.001 29 (14) 20 (22) 1.6 (0.9–3.1) 0.12 22 (11) 27 (30) 3.4 (1.8–6.5) < 0.001 No 194 (92) 69 (85) 1 184 (92) 79 (86) 1 189 (94) 74 (81) 1 Yes 17 (8) 12 (15) 1.9 (0.9–4.4) 0.084 16 (8) 13 (14) 1.9 (0.9–4.1) 0.10 12 (6) 17 (19) 3.6 (1.6–7.9) 0.001 No 158 (75) 63 (78) 1 151 (76) 70 (76) 154 (77) 67 (74) 1 Yes 53 (25) 18 (22) 0.9 (0.5–1.6) 0.61 49 (24) 22 (24) 0.9 (0.5–1.7) 0.91 47 (23) 24 (26) 1.2 (0.7–2.1) 0.58 No 162 (77) 52 (64) 1 148 (74) 66 (72) 1 157 (78) 57 (63) 1 Yes 49 (23) 29 (36) 1.8 (1.1–3.2) 0.030 52 (26) 26 (28) 1.1 (0.6–1.9) 0.685 44 (22) 34 (37) 2.1 (1.2–3.7) 0.006 No 124 (59) 26 (32) 1 108 (54) 42 (46) 1 121 (60) 29 (32) 1 Yes 87 (41) 55 (68) 3.0 (1.8–5.2) < 0.001 92 (39) 50 (54) 1.4 (0.9–2.3) 0.19 80 (40) 62 (68) 3.2 (1.9–5.5) < 0.001
P values were obtained using Pearson's χ
Using IHC, 142 patients (49%) did not show any VI. 69 patients (24%) showed LVI only, 58 (20%) showed BVI only whereas 23 (8%) showed both LVI and BVI, giving overall frequencies of 32% (n = 92) and 28% (n = 81), respectively. Of the 201 patients (69%) classified on HAS sections to be VI negative, IHC classified 48 to have LVI and 25 to have BVI, indicating that 22% (n = 66) of cases previously reported to be negative showed VI by IHC. Of the 91 cases (31%) classified as VI positive on HAS sections, IHC could not confirm this in 6 cases (7%).
BVI and LVI were not significantly associated (OR 0.8, CI 0.5–1.4, p = 0.48). (Supplementary Table 1, additional file 1).
Table 2 shows the associations between clinicopathological variables and different categories of vascular invasion assessed with and without IHC. BVI was associated with higher age at cystectomy (OR 1.8, CI 1.1–3.1, p = 0.022), higher pathological stages (OR 5.8, CI 2.9–11.7, p < 0.001), positive lymph nodes (OR 3.5, CI 1.9–6.3, p < 0.001), bladder neck involvement (OR 4.3, CI 2.3–8.1, p < 0.001), local recurrence (OR 1.8, CI 1.1–3.2, p = 0.030) and distant metastases (OR 3.0, CI 1.8–5.2, p < 0.001). LVI was associated with positive lymph nodes (OR 2.6, CI 1.5–4.5, p < 0.001) and micropapillary divergent differentiation (OR 2.5, CI 1.0–6.2, p = 0.042).
VI assessed with and without IHC were significantly associated for both BVI and LVI, BVI showing the strongest association (OR 11.3, CI 6.2–20.4, p < 0.001, and OR 2.9, CI 1.8–5.0, p < 0.001, respectively). (Supplementary Table 2, additional file 1).
When including BVI and LVI in a multivariable logistic regression model adjusted for relevant clinicopathological variables, (gender, age, pathological stage, micropapillary divergent differentiation, neoadjuvant chemotherapy) both BVI and LVI independently predicted regional lymph node metastases (OR 2.7, CI 1.4–5.2, p = 0.003 for LVI and OR 2.4. CI 1.2–4.8, p = 0.018 for BVI) (Supplementary Table 3, additional file 1).
Including BVI and LVI in a multivariable logistic regression model adjusted for variables mentioned above, BVI was an independent predictor of higher pathological stages (OR 3.9, CI 1.8–8.8, p = 0.001). In contrast, the association for LVI was not significant (OR 1.5, CI 0.8–2.9, p = 0.23) (Supplementary Table 4, additional file 1). There was no interaction between BVI and LVI (regional lymph node metastases; p = 0.38. pathological stage; p = 0.70), and the impact of BVI and LVI did not change when exclusively including either of them in the model.
The observed survival is presented in Kaplan Meier curves (Fig. 2). BVI was significantly associated with reduced RFS and DSS (p < 0.001 for both). In contrast, LVI was not significantly associated with survival. When adjusting for relevant basic clinicopathological markers such as gender, age, pathological stage, lymph node status, surgical margin positivity and perioperative chemotherapy, BVI but not LVI was significantly associated with shorter RFS and DSS, as was also lymph node metastases, higher pathological stages and positive surgical margins (Table 3). These results were independent of perioperative chemotherapy.
Graph: Fig. 2 Recurrence free and disease specific survival related to BVI (A, C) and LVI (B, D). Footnotes: Survival curves are estimated by the Kaplan Meier method using the log-rank test for differences between subgroups. For each category, number of events/total number of cases are given
Table 3 Multivariable analyses of RFS and DSS of pathological variables and BVI and LVI (Cox proportional hazard method, n = 256*)
Variables Categories Multivariable analysis RFS Multivariable analysis DSS HR (95%CI) HR (95% CI) Gender Female 1 1 Male 0.9 (0.6–1.5) 0.92 1.1 (0.7–1.7) 0.82 Age 40–68 1 1 69–76 1.0 (0.7–1.6) 0.90 1.4 (0.8–2.4) 0.21 77–88 0.6 (0.4–1.2) 0.13 1.1 (0.8–1.4) 0.71 Pathological stage pT1 + 2 1 1 pT3 + 4 2.4 (1.5–3.9) 0.001 2.2 (1.3–3.7) 0.005 Nodal status N0 1 1 N1–3 2.6 (1.7–3.9) < 0.001 2.6 (1.7–4.1) < 0.001 BVI Negative 1 1 Positive 1.5 (1.0–2.3) 0.035 1.6 (1.0–2.4) 0.039 LVI Negative 1 1 Positive 0.8 (0.6–1.2) 0.36 0.9 (0.6–1.3) 0.44 Surgical margins Negative 1 1 Positive 1.9 (1.1–3.1) 0.016 1.8 (1.1–3.0) 0.027 Perioperative chemotherapy No 1 1 Yes 0.9 (0.6–1.4) 0.65 0.9 (0.5–1.4 0.57
Abbreviations: RFS recurrence free survival; DSS disease specific survival HR hazard ratio; 95% CI 95% confidence interval; BVI blood vessel invasion, LVI lymph vessel invasion. *; performed lymph node dissection
There was no interaction between BVI and LVI (RFS; p = 0.78, DSS; p = 0.73), and the impact of BVI and LVI did not change when exclusively including either of them in the model.
The 2-year observed DSS and OS for patients with BVI were 38 and 37%, compared to 73 and 69% for patients without BVI. For patients with LVI, the 2-year DSS and OS were 56 and 51%, compared to 67 and 65% for patients without LVI.
30 (18%) of the patients with negative lymph nodes showed BVI, of which 14 patients experienced relapse of disease. When analysing the pN0 subgroup, BVI was significantly associated with reduced RFS in univariable analysis (HR 2.0, CI 1.1–3.7, p = 0.0024). For DSS, we observed a trend for increased risk for BC-related deaths in the pN0 subgroup with BVI (HR 1.9, CI 0.9–3.6, p = 0.060).
VI assessed without IHC was significantly associated with poorer RFS and DSS in univariable analyses (p < 0.001) (Supplementary Fig. 1, additional file 1) When adjusting for basic clinicopathological variables, there was still a trend for increased risk of BC-related death for individuals with VI (HR 1.5, CI 0.9–2.2, p = 0.084) (Table 4).
Table 4 Multivariable analyses of RFS and DSS (Cox proportional hazard method) of pathological variables and vascular invasion assessed without immunohistochemistry (n = 256*)
Variables Categories Multivariable analysis RFS Multivariable analysis DSS HR (95%CI) HR (95% CI) Gender Female 1 Male 0.9 (0.6–1.5) 0.81 1.0 (0.7–1.6) 0.90 Age 40–68 1 1 69–76 1.0 (0.6–1.7) 0.26 1.4 (0.8–2.2) 0.22 77–88 0.7 (0.4–1.2) 0.20 1.2 (0.7–2.1) 0.52 Pathological stage pT1 + 2 1 1 pT3 + 4 2.4 (1.4–3.9) 0.001 2.1 (1.3–3.7) 0.005 Nodal status N0 1 1 N1–3 2.4 (1.6–3.7) < 0.001 2.4 (1.6–3.8) < 0.001 VI Negative 1 1 Positive 1.3 (0.9–1.9) 0.19 1.5 (0.9–2.2) 0.084 Surgical margins Negative 1 1 Positive 1.9 (1.1–3.1) 0.013 1.8 (1.1–3.0) 0.025 Perioperative chemotherapy No 1 1 Yes 0.9 (0.6–1.4) 0.60 0.9 (0.5–1.4) 0.59
Abbreviations: HR hazard ratio; 95% CI 95% confidence interval; VI vascular invasion assessed on HAS sections. *; performed lymph node dissection
This study showed 32% LVI and 28% BVI. To the authors´ knowledge, this is the largest study to date investigating LVI and BVI by IHC in cystectomy specimens. Studies in which morphological criteria to separate the vessel types have been applied have shown prevalences of LVI varying between 20.8–54.1%, and BVI between 4-29.8% [[
Former studies [[
In this study, we found that BVI has a stronger association with recurrence and reduced survival than LVI. There is a paucity of studies using IHC in this field. Of note is the work of Afonso et al. [[
Our findings might indicate that some tumors are inclined to direct hematogenous spread. This effect could explain why BVI remains an independent prognostic factor in multivariable survival analyses. Notably, studies of disseminated tumor cells from the bone marrow indicate that hematogenous spread is often an early event in tumor progression [[
VI detected without IHC is considered a strong prognostic factor. However, in our study, when adjusting for basic clinicopathological variables we did not find this feature to be significant in multivariable RFS and DSS survival analyses, although we observed a trend for increased risk of BC-related death for individuals with VI detected on HAS sections. Similar results have been described in other studies [[
As for tumors located in the bladder neck area, our findings are in line with previously reported associations between VI assessed by H&E and bladder neck involvement [[
Immunohistochemical assessment revealed a greater variation in blood vessels regarding size. In many specimens, BVI was observed in small vessels morphologically indistinguishable from small lymph vessels, or retraction lacunae in some cases. We believe that the observed higher sensitivity in detecting vessel invasion by using IHC partly can be explained by the facilitated detection of BVI, in particular, in small venules. We also noted that CD31 positivity might be scarce in blood vessels of larger size.
We recognize that our study have some limitations. The study is retrospective in design, spanning 18 years, without standard procedures of the sampling of neither the cystectomy nor lymph node specimens. There are probable changes in surgical and oncological practice as well as inherent biases in the study cohort that could have impact on outcome. In particular, the exclusion of ypT0 cases due to study design will have affected both the observed rate of neoadjuvant therapy in the cohort as well as it may explain the estimated lack of impact of neoadjuvant therapy when adjusting for this factor in the survival analyses. On the other hand is the follow up comprehensive and complete.
Of the 91 cases classified as VI positive on HAS sections in our study, IHC could not confirm this in 6 cases (7%). This could be due to misinterpretation in cases where artificial tissue shrinkage might have given the impression of vascular spaces. A contributing factor might have been the cutting of deeper sections when performing IHC. The choosing of a single section of the total cystectomy specimen to perform IHC could further imply that other areas of BVI and LVI might have been missed. Most cases of LVI visualized by D2–40 were apparently easy to interpret, although some staining in myofibroblastic cells might represent a source of misclassification towards false positive assessment, as D2–40 previously has been shown to be a marker for urinary bladder myofibroblasts [[
Even though LVI was found to be associated with several aggressive tumor features, and in particular demonstrated the ability to independently predict regional lymph node metastases, BVI indicated a poorer prognosis. Our findings concur with the general view that BVI is associated with more widespread metastases to distant organs. In survival analyses, BVI was significantly associated with shorter RFS and DSS whereas LVI was not. In conclusion, our study has shown that in invasive BC, lymphatic involvement is more frequent than BVI, but blood vessel involvement is a stronger prognostic factor, although more studies need to confirm our observations. Furthermore, associations between blood and lymph vessel invasion and BC molecular subtypes ought to be clarified in future studies. The importance of distinguishing blood vessel invasion from lymphatic invasion in the pathology report is implied.
We gratefully thank Mrs. May Lisbeth Plathan, Dr. Gunnar Snersrud, Mrs. Hege Jorgensen and bioengineers at Vestfold Hospital Trust for their excellent technical assistance.
BC, TAK and ESH contributed to study design, data collection, data interpretation and literature search. BC, TAK, ESH and BKA performed data analyses, generation of figures and contributed to writing the manuscript. All authors approved the final version of the manuscript.
This work was supported by The Vestfold Hospital Trust Research Fund (project no.198218).
All data generated or analysed during this study are included in this published article and its supplementary information files.
Study participants submitted broad, informed consent, and data were de-identified for the analyses. The study was approved by the Regional Ethics Committee of South Eastern Norway (REK 2019/532).
Not applicable.
The authors declare that they have no competing interests regarding this paper.
Graph: Additional file 1: Table S1. Associations between LVI and BVI assessed by IHC. Table S2. Associations between VI assessed by HAS and by IHC. Table S3. Associations between lymph node metastases and various clinicopathological variables (logistic regression). Table S4. Associations between higher pathological stages and various clinicopathological variables (logistic regression). Fig. S1. Kaplan Meier curves RFS (A) and DSS (B), VI assessed without IHC. Fig. S2. Kaplan Meier curves RFS (A) and DSS (B), different categories of VI assessed by IHC. Footnotes: Survival curves are estimated by the Kaplan Meier method using the log-rank test for differences between subgroups. For each category, number of events/total number of cases are given.
• VI
- Lymphovascular invasion
• LVI
- Lymph vessel invasion
• BVI
- Blood vessel invasion
• IHC
- Immunhistochemistry
• HAS
- Hematoxylin-azophloxine-saffron
• RC
- Radical cystectomy
• RFS
- Recurrence free survival
• DSS
- Disease specific survival
• BC
- Bladder cancer
• OS
- Overall survival
• OR
- Odds ratio
• CI
- Confidence interval
• HR
- Hazard Ratio
• IQR
- Interquartile range
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