Aims To assess venous invasion (VI) and its relation to distant metastases in colorectal cancer (CRC).
Methods Primary untreated CRC cases were assessed for VI. All tumour blocks were stained with H&E and orcein. The presence of VI and nodal status were then correlated with the presence of synchronous or metachronous distant metastases.
Results VI was detected more frequently with the orcein stain (18% versus 71%). Eleven tumours (nine node-positive tumours, all VI positive) were associated with synchronous distant metastasis. During a median follow-up of 17 months nine further cases were diagnosed with distant metastasis (six node-positive tumours, all VI positive). The specificity and sensitivity of the presence of nodal metastasis for predicting distant metastasis were 0.56 and 0.75, respectively. The same values for orcein-detected VI were 0.39 and 1, respectively.
Conclusions Elastic stains such as the orcein stain enable the detection of clinically relevant VI with greater frequency than conventionally stained histological slides. If nodal involvement is an indication for systemic chemotherapy, the data presented here suggest that VI detected by the orcein stain should also be an indication for systemic chemotherapy.
- Colorectal cancer
- colorectal carcinoma
- elastic stain
- venous invasion
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Colorectal carcinoma (CRC) is among the leading causes of cancer related mortality, and mortality is related to distant metastases. Prognostic factors of the disease include the depth of tumour invasion through the layers of the bowel wall, lymph node involvement and the presence of distant metastases.1 Extramural venous invasion (VI) has also been recognised as a prognosticator of CRC,2 3 but its reported incidence shows a rather wide variation.4–6 The presence of vascular invasion is an obvious prerequisite for the development of blood-borne metastases. Although vascular invasion may certainly involve capillaries, lymphatic and blood capillaries cannot be reliably differentiated from each other on conventionally stained slides. Immunohistochemistry to demonstrate relatively specific lymphatic endothelial markers might be useful in distinguishing between lymphatic and blood capillary involvement,7 but it is impractical for routine practice. VI is distinct from lymphatic invasion and is associated with the development of distant metastases.8–10 Elastic stains have been implemented in the detection of this phenomenon, as they result in a higher rate of visualisation.11–15 Endothelium specific immunostains may also increase the detection rate of vascular invasion,13 but help identifying VI less often, because the endothelium may disappear from tumour-invaded vessels.16 17 Not only extramural, but also intramural, VI has been found to be associated with the presence of metastasis4 15; therefore, the localisation of VI is probably less important than its presence.
Although several studies have assessed the incidence of VI with the routine use of an elastic stain, and the presence of VI has also been correlated with distant metastases, to our knowledge, no studies have tried to evaluate the association of VI with metastases occurring later during follow-up and compare VI with nodal status in this respect. The present study aims to do this in a series of patients with CRC.
Materials and methods
CRC resection specimens received at the Department of Pathology in 2007 were retrospectively collected from an institutional database. The tumours were staged according to the TNM (tumour, node, metastases) classification.18 19 Besides the classification into pT categories reflecting the depth of infiltration or invasion of the peritoneum or nearby organs, and pN categories reflecting the nodal status, the initial M categories for the presence or absence of distant metastases were derived from data available at the multidisciplinary meetings. For the initial M categories, intraoperative biopsy and histology proven metastases (pM) and clinically detected metastases (M) were considered. For the initial staging, patients were evaluated routinely by chest radiograms and abdominal ultrasound, and further imaging studies were performed when required. During follow-up, the M category evaluation was also complemented by autopsy-derived data and patients' charts were used as source.
All tumour blocks were prospectively and routinely assessed for VI in slides stained with orcein. Briefly, after deparaffinisation, the slides were kept overnight in a solution of 0.1 g orcein (Reanal, Budapest, Hungary) in 100 ml 70% ethanol and 2 ml concentrated hydrochloric acid. This was followed by differentiation in 70% ethanol, dehydration, clearing in xylene and mounting. First the H&E-stained slides were assessed for VI, and this was followed by the examination of the synchronously stained orcein slides. The results on VI were reported as detected on H&E or orcein. The tumours were therefore also categorised according to the V classification of the TNM: V1 for the presence of microscopically detected VI, and V0 for its absence.
Follow-up data were available up to April 2009. The following cases were excluded from the analysis: intramucosal carcinomas with no invasion beyond the muscularis mucosae (pTis according to the TNM classification2 18 19), recurrent cancers, cases treated with neoadjuvant therapies before histopathological evaluation, cases with synchronous or metachronous cancers elsewhere in the body, and Mx cases due to either lack of follow-up data or postoperative death without autopsy.
The association of VI or nodal involvement and distant metastases was evaluated with the Fisher exact test (VassarStats, Vassar College, Poughkeepsie, New York, USA). Significance level was set at p<0.05 (two tailed).
Due to the retrospective nature of the study and the lack of specific patient identifiers among the data analysed, according to local regulations, no institutional ethical committee approval was necessary. However, an institutional data safety manager approval was needed and obtained.
After the exclusion of the cases mentioned in Methods, 89 patients (47 men and 42 women) remained for analysis. These included 16 patients with rectal tumours and 73 patients with colon tumours. The median number of orcein-stained slides was six. VI was detected in 16/89 (18%) cases on H&E-stained slides, whereas it was found to be present in 63/89 (71%) cases after the evaluation of orcein-stained slides (figure 1). VI was more common with greater pT categories and was also more common with nodal involvement; all tumours classified as pN2 had VI. The initial staging results are shown in table 1. Eleven cases had distant metastases at the initial staging. The median number of lymph nodes examined was 18 (range 4–55). There were 46 (52%) node-positive cases, nine of the M1 tumours were among these, whereas two metastatic cases belonged to the node-negative patients.
The lymph node status (pN0 versus node positive) nearly showed an association with an initial M1 status (p=0.05). VI detected by H&E was not associated with an initial M1 status (p=1), but VI detected by the orcein stain showed a significant association with synchronously detected distant metastasis (p=0.029).
Ten M0 cases with less than 6 months of follow-up after surgery were excluded from subsequent analyses. Of the remaining 79 tumours 14 (18%) demonstrated VI on H&E slides and this number rose to 56 (71%) after the analysis of orcein-stained slides. Chemotherapy was administered to 45 patients (30 with VI and nodal involvement, 11 with VI alone, three with nodal involvement alone, and one with no VI or nodal metastasis) and no systemic therapy was given to 34 patients (six with VI and nodal involvement, eight with VI alone, one with nodal involvement alone, and the remaining 19 with no VI or nodal metastasis). For the 68 cases that were initially M0, the median follow-up time was 17 months (range 6–28 months); during this period nine patients with colon cancer developed distant metastases detected after 4–22 months(median 9 months) following surgery (table 2).
Five of the 20 finally metastatic cases were detected in the node-negative group; all the 20 metastatic cases had VI detected by the orcein stain.
Synchronous or metachronous distant metastases were associated with lymph node involvement and VI detected by orcein (p=0.02 and p=0.001, respectively), whereas H&E-detected VI showed no association (p=0.31).
Considering the presence of VI or lymph node metastasis as predictors of distant metastasis (either detected at the time of initial staging or during follow-up), the predictive values of VI detected by H&E or orcein and nodal involvement are shown in table 3.
The presence of lymph node metastasis is often considered as an indication for adjuvant chemotherapy in colon cancer.20 Nowadays there are also other factors (eg, poor differentiation, lymphatic/vascular invasion, less than 12 nodes assessed) that would also result in the consideration of such a treatment in node-negative colon cancer, although observation is also an alternative for some of these patients.21 For rectal cancer, lymphatic/vascular invasion is also recognised as an unfavourable histological feature with some impact on the recommendation of adjuvant therapy.22
Even the presence of one metastatic lymph node results in worse outcome, and the worsening of prognosis increases with the number of lymph nodes involved.2 Interestingly, the qualitative nodal status (positive versus negative) just failed to show a significant association with synchronous distant metastases, although it was associated with the presence of synchronous or metachronous metastases detected after a median follow-up of 17 months.
VI demonstrated by H&E was less common than VI demonstrated by orcein, and statistically showed no association with distant metastases; this is in keeping with data from Erlangen.23 In contrast, VI demonstrated by the orcein elastic stain was significantly associated with the development of synchronous or metachronous distant metastasis. These results strengthen the value of routine elastic stains for the demonstration of VI, as suggested by several previous publications.9–13 The Royal College of Pathologists' current dataset recommendation suggests the use of elastic stains to confirm or reject the presence of VI in suspicious cases,24 but the routine use of elastic stains may highlight VI even in cases where there is no suspicion on H&E-stained slides. As reporting (extramural) VI is part of most current recommendations,24–26 its enhanced detection can also be reasonable to recommend.
Although vascular invasion is required for the development of haematogenous dissemination, its presence cannot be equated with the presence of metastases. First, cases with VI might not obviously develop blood-borne metastasis, as tumour cells may not always be transported from an occluded vessel to the distant site, and also because the metastatic process is complex and requires more than the presence of tumour cells in a vessel. These cells need to seed, extravasate and grow at the distant site. Second, vascular invasion may occur at the capillary level, and this is more difficult to detect and differentiate from lymphatic invasion, as mentioned above. Third, the demonstration of VI is a random statistical event. As histopathology is based on sampling, VI might be present but missed by the slides examined. For its identification on histological slides, VI must be extensive or if non-extensive, be recognised by chance. Besides this spatial relationship, there is also a temporal relationship. Following invasion of tumour cells into the vascular lumen, floating-type VI may vanish as the tumour cell emboli leave the site of origin. These considerations should explain why metastases may form without identified VI. Indeed, VI has been detected in only 70–90% of M1 cases.4 9 15 In contrast to the cited series, VI was seen in all cases initially staged as M1 or developing metastases during a short follow-up. It would be too optimistic to believe that, with careful examination, an elastic stain may identify all patients having VI, but for sure, it helps to disclose more VI-positive cases than the conventional H&E stain. The presence of VI on orcein-stained slides is associated with the development of distant metastases with a positive predictive value of 36%, which is very similar to the positive predictive value of nodal involvement. Such a comparison between nodal status and VI was not attempted in similar studies dealing with the relation of VI and distant metastases in CRCs.4 8 9 15
Comparison of VI (demonstrated by orcein) versus nodal status shows that although both are predictive for the development of distant metastases, the better specificity of the first is compensated by the better sensitivity of the other. Although VI often occurs in patients with nodal involvement, its presence alone (without nodal involvement) is sufficient for the development of distant metastasis. Therefore, if nodal status is an indication for systemic treatment, VI might also be considered as such. In fact, the presence of VI in Dukes B CRC has been found to be associated with worse survival than the presence of a single metastatic lymph node, and therefore the consideration of chemotherapy for these patients is obvious.27 VI detected by elastic stains might also be an important feature in Dukes A CRC.10
Our study has some limitations that need to be mentioned. First, the follow-up time was rather short, but 2007 was the first full year with data on VI detected by orcein. Second, the majority of the tumours analysed were from the colon, as preoperative treatment was an exclusion criterion, and likewise all patients with metachronous metastasis had primary colon cancer.
Orcein staining is an easy and cheap method for detecting VI in CRC. Our data show that its results correlate with the presence of synchronous metastases or the development of metastasis at any time, although the follow-up was short in this series. Therefore, we suggest that orcein or another elastic stain should be routinely used for the evaluation of CRC, at least for the node-negative and H&E-based VI-negative cases. VI detected by this simple method should be seriously considered as a factor in favour of giving systemic treatment.
Venous invasion (VI) is demonstrated with increased frequency in colorectal carcinoma with the orcein elastic stain when compared with H&E staining.
VI demonstrated by orcein is associated with synchronous or metachronous distant metastasis similarly to lymph node metastasis.
Lymph-node-negative patients with VI may require adjuvant systemic treatment on the basis of the latter phenomenon.
Competing interests None.
Ethics approval This study was conducted with the approval of the Bács-Kiskun County Teaching Hospital IRB.
Provenance and peer review Not commissioned; externally peer reviewed.