3. Discussion
The neoplasms reported in this study comprising those revised from the literature, displayed peculiar rhabdoid features and were associated with an ominous clinical course with a large majority of patients surviving for less than six months. The diagnostic hallmark of these neoplasms is the presence of rhabdoid cells characterized by round eosinophilic aggregates of intermediate filaments that displace the nucleus to the cell periphery. Other consistent morphologic features are: The non-cohesive growth of tumor cells, the scarcity of tumor stroma, the abundance of tumor infiltrating neutrophils, and the scarcity of lymphocytic infiltration. The main immunohistochemical findings are: The expression of vimentin and pancytokeratin within filamentous cytoplasmic inclusions, the loss of membranous E-cadherin, the nuclear dislocation of β-catenin, the lack or reduced expression of important markers of colonocyte differentiation such as CK20 and CDX2, the marked nuclear p53 accumulation, and the high proliferative Ki-67 index.
The majority of CRbCs consist solely of rhabdoid cells and are indicated as “pure”, while other CRbCs combine a rhabdoid component with an adenocarcinoma component most frequently focal and confined to the tumor periphery and are designated as “combined”. The presence of a transitional zone in combined CRbCs with a continuum between rhabdoid and non-rhabdoid cells indicates that rhabdoid cancers cells (RbCs) might have originated from dedifferentiated primary colorectal cancer [
5,
28].
The most relevant immunohistochemical finding for the diagnosis of CRbC is the coexpression in tumor cells of pancytokeratin and vimentin. This was found in all CRbCs but not in PDMCs examined. Coexpression of a mesenchymal marker such as vimentin and epithelial markers is one of the phenomena that characterize the process of epithelial–mesenchymal transition (EMT) [
29]. This is a unique process in which cells lose epithelial features and acquire mesenchymal qualities [
30]. The process of EMT is characterized by the reduction of epithelial markers and increase of mesenchymal markers [
31]. E-cadherin is the most important mediator of cell adhesion in epithelial tissues and loss of E-cadherin is a crucial step in EMT. During EMT, loss of E-cadherin is associated with the release of β-catenin, which is consequently translocated to the nucleus where it activates the WNT signaling pathway [
32]. In colorectal cancer, altered expression of E-cadherin and β-catenin and progressive increase of vimentin in late stages are associated significantly with aggressive tumor cell behavior and, furthermore, confer resistance to cancer drugs [
33,
34]. In addition, in a recent study [
35], it has been demonstrated that the gene expression profile of tumor budding regions in CRC closely matches that of consensus molecular subtypes 4 (CMS4) (mesenchymal) subtype, while the bulk presents a CMS2 (epithelial profile).
Previous immunohistochemical results demonstrating loss of membranous E-cadherin in CRbC [
2,
3,
12] and our results demonstrating β-catenin nuclear localization and loss of colonic epithelial markers such as CK20 and CDX2 support the pathogenetic involvement of EMT as an essential player in the heterogeneous make-up of CRbC. In addition, the loss of membranous E-cadherin and β-catenin suitably explains the discohesive histologic pattern of CRbC.
Cells undergoing EMT maintain the same genomic background in both mesenchymal and epithelial states, but during the progression of EMT, the gene expression profile significantly changes. A series of protein complexes, known as chromatin remodelers, are crucial to mediate this event as they can slide, destabilize, or relocate nucleosomes in an ATP-dependent manner [
36]. The SWI/SNF mating-type switching (SWI) and sucrose nonfermenting (SNF) subfamily has specifically been investigated in malignant rhabdoid tumors, pediatric and highly lethal neoplasms of the kidney and brain where
SMARCB1 (INI-1) is frequently mutated either at germline or at somatic level [
37]. To date, the role of
SMARCB1 (INI-1) inactivation remains to be determined in CRbC and only few studies reported SMARCB1 (INI-1) immunohistochemical loss in a small subset of CRbCs that were frequently
BRAF-mutant, MSI, and CIMP [
1,
3,
11]. These data allowed to hypothesize that
SMARCB1 (INI-1) may occur as a secondary molecular event during EMT in a subset of CRCs characterized by
BRAF V600E mutation, MSI, and CIMP, virtually conferring a rhabdoid phenotype [
2]. In line with this hypothesis, Wang et al. [
10] demonstrated that loss of
SMARCB1 (INI-1) expression occurs at least focally in 0.46% of 3051 CRCs and is associated with higher grade, larger tumor size, poorer survival, MSI, and
BRAF V600E mutation.
In this context, our study sheds some light on the biological features of this rare entity thanks to a genetic/epigenetic comparative analysis of CRbCs and PDMCs showing BRAF V600E as a common prominent genetic feature. A first important finding of our analysis was that CRbC mainly included BRAF mutant/MSS cancers without CIMP. By contrast, PDMC only comprised BRAF mutant/MSI and CIMP cancers. Two BRAF mutant/MSI cases were observed among CRbCs and in one case we could exclude CIMP. Interestingly, both these cases showed a reduced SMARCB1 (INI-1) expression but not a complete loss of the protein as we found in the remaining five CRbCs.
To date, due to their rarity, the
BRAF mutant/MSS colorectal cancers have not been as thoroughly studied as
BRAF/MSI cancers. Although both subsets derive from a serrated polyp due to the presence of the
BRAF mutation and are clinically associated with a detrimental patient outcome,
BRAF mutant/MSS colorectal cancers diverge from
BRAF/MSI cancers with the development of clinicopathologically and genetically distinct aberrations [
27]. Histologically,
BRAF mutant/MSS cancers show more adverse morphological features compared with
BRAF/MSI cancers, such as frequent tumor budding; high-grade neuroendocrine carcinomatous component; a lack of tumor infiltrating lymphocytes; frequent lymphatic, perineural, and venous invasion; and increased lymph-node metastases [
38,
39,
40]. In addition, it is of interest to recall that in one study [
41]
BRAF mutant CRCs on the basis of gene expression have been split in two subtypes called BM1 and BM2. The subtypes displayed differences in overall survival (OS) and progression free survival (PFS). The enrichment of BM1 group in EMT signature and CMS4 consensus subtype correlates with poor survival of patients.
For the first time, with this work, we suggest that
BRAF mutant/MSS cancers include the rare entity of CRbCs, characterized by a strong activation of EMT and complete loss or reduced expression of SMARCB1 (INI-1). Moreover, a recurrent finding of CRbCs in this study, was the abundance of tumor-infiltrating neutrophils which contribute to the formation of the tumor microenvironment. Although neutrophils were at first considered to possess defensive functions against cancerous cells, it has been demonstrated that some subtypes of neutrophils, known as tumor-associated neutrophils (TANs) possess a tumor-supporting function [
42]. TANs contribute to tumor invasion and angiogenesis through production of matrix metalloproteinases, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). Interestingly, intratumoral neutrophils in CRCs have been found to correlate closely with a malignant phenotype and to represent an independent factor of poor prognosis for the patients [
43].
Molecularly,
BRAF mutant/MSS cancers have multiple genetic aberrations that are representative of typical changes associated with both serrated and conventional pathways. Although they display hypermethylation events that commonly characterize all
BRAF mutant cancers, this subset of tumor shows lower frequency of CIMP than
BRAF/MSI cancers [
44,
45]. In line with this observation, CIMP was not found in CRbC in contrast to PDMC, analyzing the conventional panel of genes suggested to identified CIMP in tumors of the serrated pathways. Although this result does not preclude the presence of gene hypermethylation in CRbCs, the use of this gene panel may be useful to distinguish them from tumors of the classical serrated pathway.
Finally,
TP53 mutation has been correlated with advanced stages and with conventional pathway in CRCs.
BRAF mutant/MSS cancers have been found to have a comparably high rate of
TP53 mutation as the
BRAF wild-type cancers, whereas
BRAF mutant/MSI were confirmed to have a low rate of mutation [
44]. In our study, all but one tumor showed
TP53 mutation and no specific differences we observed comparing CRbCs and PDMCs. An interesting observation was that
TP53 mAFs were often higher than
BRAF mAF in most of the tumors analyzed. These data emphasize a driver role of
TP53 in the early phases of the development of these tumors suggesting that in addition to the constitutive activation of the MAP kinase pathway through
BRAF/RAS mutations, simultaneous upregulation of anti-apoptotic pathways may be crucial for the rapid and aggressive growth of these tumors.
Rhabdoid carcinomas seem to be resistant to conventional therapy used for gastrointestinal neoplasms (FOLFOX, FOLFIRI scheme associated with monoclonal antibody). Moreover, anthracycline based regimes generally used in sarcoma do not seem effective. The co-presence of BRAF and P53 mutations in CRbCs suggests the possible therapeutic role of a double block acting on BRAF and p53.
In summary, CRbCs are characterized by BRAF and less frequently KRAS mutations co-occurring with TP53 mutations. Coexpression of pancytokeratin and vimentin, dense neutrophilic infiltration, loss/reduced expression of nuclear of SMARCB1/INI, and low frequency of CIMP are useful markers to recognize these rare aggressive tumors. Elucidation of the genetic and epigenetic landscape alterations of these tumors is crucial to hypothesize specific treatments with novel biological agents such as MAPK inhibitors and small molecules blocking p53 degradation and epigenetic drugs.