In the present study, we investigated whether tumor budding is a prognostic factor in patients with rectal adenocarcinoma treated with neoadjuvant therapy. Our results showed a strong connection between posttreatment budding and a more aggressive tumor biology, i.e., correlation with adverse clinicopathological features, such as deeper tumor infiltration or a higher frequency of lymph node metastases. Irrespective of the staining method used, patients with tumor budding had a significantly worse prognosis for disease-free survival and overall survival. These aspects have already been described in patients with chemotherapy-naïve colorectal cancer [4,5,6,7,8,9] and included as a recommendation in major national guidelines for the assessment of early invasive cancer [11, 13, 14].
Budding has been described as a prognostic feature after chemoradiotherapy in rectal cancer patients in several publications with the general limitation of a retrospective study design. In previous studies, budding was reported in 10.1–63.2% of cases due to different methodologies used for evaluation [3, 15,16,17,18,19,20]. Budding has been shown to be a negative prognostic factor for survival in different kinds of study designs and for a broad range of cut-offs. However, most of the previous studies could demonstrate effects on survival only in univariate analysis or limited to disease free survival [15,16,17,18,19]. Including patients with complete response in the analysis appeared to attenuate the prognostic impact of tumor budding. In our opinion, it is self-evident that budding cannot be evaluated in patients with a complete response. Therefore, in our study, we focused on cases with poor response in order to stratify the outcome of patients with residual tumor burden. By this approach, we were able to demonstrate a strong impact on disease free survival and overall survival in univariate and multivariate analysis.
Of the most recent studies, Jäger et al. [3] can be compared to our own study. As in our study, they evaluated budding not only at the invasive front but also throughout the tumor. The high budding rate of 63.2% compared to our results can be explained by the low cut-off of two buds in one microscopic field, whereas in our study a cutoff of 5 buds was used according to standard criteria of Ueno et al. [10]. As in our study, budding remained a significant parameter in multivariate analysis for disease free survival. However they failed to demonstrate this for overall survival, presumably, because patients with complete response were included in the statistical analysis.
Only one previous study claimed that a single cell pattern of growth in the invasive front was a prognostic factor for prolonged cancer-specific survival [21]. They interpreted the single-cell growth pattern as an indicator of tumor cell regression. However, they did not evaluate budding as a standardized parameter but rather as a semiquantitative score of the tumor growth pattern. Furthermore, patients with a complete pathological response were included in the survival analysis, undermining the impact of budding as a parameter for the stratification of patients with a poor response.
In our study, immunohistochemical staining showed that budding had a considerable prognostic influence and was even superior to that of conventional parameters such as ypT and ypN stage, which have been used in routine so far. Therefore, the assumption arises that the assessment of posttreatment budding may improve the commonly used TNM classification for stratifying rectal cancer patients treated with neoadjuvant therapy and for predicting prognosis.
However, there is still a general lack of a unified definition of tumor budding. At the International Tumor Budding Consensus Conference (ITBCC) in 2016, a consensus for a standardized definition of budding and for an evaluation method was reached, but only for colorectal cancers without neoadjuvant chemoradiotherapy so far [22]. Tumor budding was defined as a single cell or a cluster of up to four tumor cells assessed in one hotspot measuring 0.785 mm2 at the invasive front. Furthermore, a three-tier system was recommended with a whole budding count to allow adequate risk stratification. The documentation of tumor budding after neoadjuvant therapy has not yet been suggested for daily diagnostic practice because of data gaps in its prognostic value in treated rectal cancers as well as a lack of a standardized evaluation method for these cancers.
The initial morphology of the tumor is often modified after chemoradiation, with phenomena such as heavy fibrosis, breaking up of the glandular tumor structures and necrotic areas. Due to these factors, the assessment of budding proposed by the ITBCC becomes challenging. It should also be mentioned that after neoadjuvant therapy, tumor borders may appear fragmented, so the tumors occasionally form several invasive fronts in the context of fibrosis and inflammation. Owing to these histological changes, we assessed tumor budding not only at the utmost invasive front (such as in cancers without chemoradiation) but also in-between invasive foci. Lugli et al. [23] and Rieger et al. [24] showed that intratumoral budding in chemotherapy-naïve patients with colorectal cancer is generally associated with peritumoral budding. They found that as long as the observer investigates the densest region with budding, it does not matter whether buds are detected at the invasive front or within the tumor. Our method used to assess budding without being limited to the invasive front in neoadjuvant-treated cancers was fundamentally based on those findings. With our method, we were able to address the abovementioned problems occurring after preoperative therapy while keeping the method relatively simple and potentially reproducible for other observers.
For our budding analyses, we used the one hotspot method, as recommended by the ITBCC and originally proposed by Ueno et al. [10]. It is a fast and simple way to subdivide patients into two different categories that are prognostically highly relevant. Even in patients with little residual tumor after preoperative therapy, the method was able to find high-risk patients. Although the cut-off was set to merely 5 buds per hotspot, as proposed for the stratification of pT1 carcinomas in polyps [10], it was possible to apply the same cut-off for locally advanced cancers, with a resulting high impact of both disease-free survival and overall survival.
In addition to H&E staining, we used IHC staining to make buds more readily visible. Kai et al. [25] were able to show that IHC can reduce interobserver variability in the evaluation of budding between unskilled observers. This would make IHC suitable for training pathologists who are inexperienced in this field. As described in previous studies for colorectal cancers without prior chemotherapy, cytokeratin staining detected more budding-positive cases [26]. In our study, we detected more budding-positive cases by the means of IHC staining as well, and this method improved the prognostic value of tumor budding assessment. IHC helped to stratify patients into even more meaningful risk groups than H&E staining. When analyzed with IHC staining, fewer budding-negative cases were found, and these had a better prognosis than budding-negative cases found by H&E (five-year disease-free survival rate: 87% vs 75%; and five-year overall survival rate: 92% vs 84%). The employed cut-off may therefore identify patients with a favorable prognosis who might be able to refrain from adjuvant therapy. If the cut-off for budding on IHC was higher, high-risk patients would more likely come to light. In these patients, more intensive aftercare might be recommended. The ideal cut-off for the evaluation of budding in IHC-stained sections still needs to be investigated. In tumors without neoadjuvant therapy, the ITBCC recommends the use of IHC in difficult cases (such as for distinguishing buds from peritumoral inflammation reaction), but the final evaluation should still be performed on H&E-stained slides [22]. In the case of pretreated tumors, the role of IHC and H&E staining still needs to be determined. Since, for example, more tumor inflammation occurs in such cases, IHC might play a more central role in the evaluation of these tumors. So far, only two previous studies regarding IHC staining in posttreatment budding exist [17, 18]. Concerning this issue, more investigations should be performed.
The main limitation of this work is its retrospective nature. It also needs to be determined whether the number and composition of included patients may vary among patients with different ethnic backgrounds. In addition, the recorded number of events during the follow-up could be a limitation as well, especially in the interpretation of the Cox regression models due to the possible lack of outcome data. Nevertheless, current data on the prognosis of tumor budding in rectal cancers treated with neoadjuvant therapy are unsatisfactory.
It is remarkable that lymph node status did not have a significant effect on outcome in our multivariate survival analysis. In fact, previous studies of neoadjuvant therapy in rectal cancer found strong associations of lymph node status with survival [27, 28]. However, in these studies, patients with complete pathological response were included in the analysis, who were here excluded due to being non-informative for tumor budding. We speculate that the specific case selection in our study may be the cause of such discrepancies.