Tumor budding assessment in CRC: why and how

“A study by colleagues in Ireland was able to demonstrate that a higher number of buds seen in a preoperative biopsy was associated with a poorer response to neoadjuvant therapy. This is the information we want to have” (Rogers AC, et al. Mod Pathol. 2014;27[1]:156–162).

“We know that budding in biopsies correlates with budding in the resection specimen, and that budding in biopsies predicts lymph node and distant metastases, as well as patient survival,” Dr. Dawson says. “But it’s going to be tough to get this implemented because there are going to be a lot of questions in terms of quality measurement that need to be addressed.

“For instance, how many biopsies are going to be needed? How much invasive tumor do we need? How deep do the biopsies need to be taken, et cetera?” And most pathologists know that diagnosing invasive colorectal cancer on biopsies can sometimes be challenging, she notes. “So these are things that will definitely need to be addressed by subsequent studies before we use budding in biopsies.”

In speaking about the biology of tumor budding, Dr. Dawson says tumor buds have different protein expression profiles than the main tumor body. “And it appears that tumor buds show overexpression of markers related to epithelial-to-mesenchymal transition, cell migration and cell survival, and cell differentiation and cell proliferation. Ki-67, for example, is downregulated in these cells in comparison to the main tumor body.

“Most of this has to do with Wnt pathway deregulation,” Dr. Dawson says. It begins with a mutation of the APC gene and leads to the internalization in nuclear translocation of beta-catenin. This creates a complex that acts as a transcription factor causing the upregulation of genes, for instance, involved in migration. Tumor budding was recently shown to be associated with BRAF and KRAS mutations (Trinh A, et al. Br J Cancer. 2018;119[10]:1244–1251).

A study of 238 mismatch repair deficient colorectal carcinomas found that budding can be seen less in MMR-d CRC. “This probably also has to do with the lymphocyte infiltration,” she says, noting that budding-to-lymphocyte ratio could be assessed in the future. “Nevertheless, when seen in these tumors, tumor budding retains its prognostic value” (Ryan É, et al. Am J Surg Pathol. 2018;42[1]:60–68).

It would seem intuitive, Dr. Dawson says, that the high-grade tumor budding cases would fall into the consensus molecular subtype 4 mesenchymal category. A 2018 study of four cohorts had “a classifier RNA transcription profiling for all of them. And it was demonstrated that in all four cohorts, high-grade tumor budders were more likely to be of the CMS4 mesenchymal subtype. So this is the best evidence we have that high-grade tumor buds are associated with and linked to the EMT process” (Trinh A, et al. Br J Cancer. 2018;119[10]:1244–1251).

A morphological difference can also be seen in tumor buds, she says, “especially if you can visualize tumor buds with an immunostain, say a cytokeratin stain. Then you can be surprised that those start to look a bit more spindly. So that would be a morphological correlate of transitioning more toward a mesenchymal type.”

It’s well known that certain scenarios of tumor budding assessment in CRC can be challenging and might need special explanations, she says. Examples are tumors with inflammatory infiltrate, many stromal cells, angry fibroblasts, areas of glandular fragmentation, mucinous/signet cell differentiation, and after neoadjuvant therapy (Cho SJ, et al. Arch Pathol Lab Med. 2018;142[8]:952–957).

Sometimes, especially in MSI-high tumors, pathologists will see a prominent inflammatory infiltrate that can make it challenging to find the buds, Dr. Dawson warns. “A keratin stain will highlight the tumor buds, and it will make things a bit easier for you.” (Fig. 3).

Pathologists can be surprised at how many buds they see on the keratin stain that they didn’t identify on the H&E, Dr. Dawson says. “That’s okay. We know that you score far more tumor buds on a keratin stain. Then you have to go back to the H&E and count the tumor buds on the H&E.” She says they talked about this at length during the consensus conference and concluded that most of the evidence in the literature was based on H&E stains. “So that’s why we agreed to assess tumor budding on H&E stains, but with the option to do a keratin stain for orientation purposes only in difficult cases.”

Dr. Dawson also cautions that mucinous cancers sometimes produce a lot of mucin and the tumor cells become trapped in mucin pools. “The real tumor buds, the real deal, are able to migrate through tissue. The migration through tissue is what is going to get them into the vessels, the lymph nodes, the liver, the lungs and everywhere,” she says. “Tumor buds need to be in tissue and not in a mucin pool.” (Fig. 4).

In the rare instance that a colorectal cancer is only mucinous, the pathologist can say tumor budding doesn’t apply in this case and provide a comment, Dr. Dawson says. In practice, pathologists should not count tumor buds in areas of mucinous and signet ring cell differentiation and glandular fragmentation, she says.

After neoadjuvant therapy, tumor budding is not reported because it’s too hard to discriminate which cells are just residual tumor cells after therapy and which ones are the tumor buds, although some studies point toward tumor budding as an adverse marker for survival in these patients, she says. The pathologist can say that tumor budding is often not applicable in these cases (Fig. 5).

Interobserver variability is an important issue in tumor budding, Dr. Dawson says. “Typically, interobserver variability for budding has a very wide range, from fair to very good, and this all depends on multiple criteria.”

Two studies examined interobserver variability when using the ITBCC guidelines to assess and report tumor buds. The first demonstrated very poor agreement (Martin B, et al. Virchows Arch. 2018;473[2]:189–197), and there are two likely reasons, she says. “One was that they did the variability, but they did agreement for categories and not for numbers,” she says. “So the cases where somebody would say low-grade budding and the other person would say high-grade budding [were] actually very rare.”

Case mix is also a factor. “They had only very little Bd3 tumor, so most of the cases hovered around Bd1 and Bd2, and that’s when they got the high interobserver variability.” Interobserver variability for pT1 colorectal cancers tends to be higher, she says, probably because the invasive margin is so much smaller. “So people zoom into the same hotspot.”

The second study exhibited much better interobserver variability, verified on immunohistochemistry (Barel F, et al. Pathology. 2019;51[1]:46–54). Based on Kappa and intra-class correlation coefficients, good to very good interobserver agreement was obtained by analyzing vertical and lateral margins, submucosal invasion, tumor differentiation, and lymphovascular invasion. It was fair based on H&E but good using IHC.

Should pathologists do budding on IHC or on H&E? “Now this is quite conflicting in the literature. There are studies that say that IHC has better results in terms of interobserver variability, and others that say it should be done on H&E. The pro of IHC is that it highlights the buds. There are also some cons. So you have to be aware of the fact that it will be more difficult to see an individual bud or to detect a tumor cell on IHC because the nucleus is often obscured, and you don’t have the same morphology.”

Tumor budding is such a robust biomarker that it fundamentally works for nearly every type of solid cancer or carcinoma, Dr. Dawson says, pointing to an article that reported that tumor budding is associated with lymph node metastasis and poor survival in patients with esophageal and gastric intestinal-type adenocarcinoma (Berg KB, et al. Mod Pathol. 2018;31[6]:862–872). “This also has similar potential implications as in pT1 colorectal cancer.” Pathologists can use tumor budding to decide whether the patient needs a resection, or if they are okay with an endoscopic mucosal resection or an endoscopic submucosal dissection specimen, she says.

“Tumor budding is also significant for oral squamous cell carcinoma and breast carcinoma. Obviously, this is limited to ductal breast adenocarcinoma because in lobular, by definition, all cells are single tumor cells and on their own, and they don’t all qualify as being tumor buds,” Dr. Dawson says. Tumor budding has also been shown to be prognostically relevant in lung cancer, both adenocarcinoma and squamous cell carcinoma, and in urothelial cancer.

Dr. Dawson says she isn’t aware of tumor budding having been incorporated into any major reporting protocol except colorectal cancer. “So what goes beyond that is more of an experimental setting, or somebody may add it in a comment, but it’s more of a free-for-all,” she says. “We have generated a lot of studies on tumor budding in pancreatic cancer, and it stratifies wonderfully in ductal pancreatic cancers. So we report that as an additional factor that our clinicians are interested in, because they help us with our studies as well. But it wouldn’t be a factor that would tip your scale to treat the patient any differently at the moment.” 

Karen Lusky is a writer in Brentwood, Tenn.