Gastrointestinal Carcinoma with Plasmacytoid Morphology: Positivity for c-MET, Arylsulfatase, and Markers of Epithelial-Mesenchymal Transition, as Indicators of Aggressivity

Background Plasmacytoid urothelial carcinoma is a rare and aggressive histologic variant of high-grade carcinoma of the urinary bladder. Few than 250 cases have been reported in the urinary bladder till January 2019. In this paper, a case series of unusual gastrointestinal carcinomas with plasmacytoid morphology was included. Only one similar case of the stomach was previously published and no such cases were found in colon. Methods We present the complex immunoprofile, using a panel of 39 biomarkers, of the largest group of primary gastrointestinal carcinomas with plasmacytoid morphology reported in literature (one from upper rectum and six from stomach). Results All of the seven cases showed lymph node metastases and only one survived over 25 weeks after surgical excision. The indicators of aggressivity were age (over 60), advanced stage (from IIIA to IV), E-cadherin negativity, and vimentin positivity. The immunoprofile indicated unfavorable prognosis for mesenchymal-type carcinomas (negativity for E-cadherin and positivity for vimentin, with membrane to nuclear translocation or negativity of β-catenin). The survivor showed an “epithelial-type adenocarcinoma with plasmacytoid dedifferentiation”, with membrane positivity for E-cadherin and β-catenin and vimentin negativity. All of the cases expressed c-MET and were negative for HER-2. Conclusions Primary carcinoma with plasmacytoid morphology is a dedifferentiated variant of adenocarcinoma or poorly cohesive carcinomas. Vimentin positive dedifferentiated-poorly cohesive carcinomas should be considered as mesenchymal-type highly malignant carcinomas. This rare histologic variant of gastrointestinal cancer might respond to anti-c-MET tyrosine kinases.


Introduction
Plasmacytoid urothelial carcinoma (PUC) is a rare and aggressive histologic variant of high-grade carcinoma of the urinary bladder [1][2][3][4] whose diagnosis is difficult to make.
The PUC variant was described in the urinary bladder in 1991 [4] but was recognized by the World Health Organization (WHO) in 2004 [3,5].
About 1-3% of all UCs are diagnosed as PUCs [5]. 61 cases have been reported in the English literature till 2012 [3], less than 100 cases till 2017 [3], and less than 250 till January 2019. They occur more frequently in males (M:F=3:1), around the age of 69 (range 46-87 years) [4,6]. Due to their rare occurrence, few data are known about these carcinomas.
Except the urinary bladder, carcinomas with plasmacytoid morphology were also described involving other organs. Although CD138 may infrequently mark gastrointestinal carcinoma cells, only one case of primary gastric PC was reported in 2012 [7]. Another duodenal carcinoma with 2 Journal of Oncology (c) (d) Figure 1: Representative aspect of carcinoma with plasmacytoid morphology, with discohesive round to ovoid cells with eccentrically located nuclei (a-left, b-d), sometimes with nuclear pleomorphism (c). The mucinous adenocarcinoma can be associated (a-right).
plasmacytoid morphology was reported in 2017 but finally proved to be metastatic tumors from a PUC and not a primary carcinoma [3]. WHO has not yet recognized this entity as histological subtype of gastric or colorectal carcinomas [8].
Independently from the localization, carcinoma with plasmacytoid morphology is characterized by diffuse proliferation of discohesive cells with plasmacytoid morphology, with eccentrically located nuclei, indistinct nucleoli, and eosinophilic cytoplasm that express cytokeratin (CK) and the transmembrane heparan sulfate proteoglycan CD138 (Syndecan-1) in over 50% of tumor cells [3][4][5]. The tumor cells can show solid sheet-like architecture or are arranged in cords and small nests [3].
In this paper, we present a comprehensive immunoprofile of 7 primary carcinomas with plasmacytoid morphology of the gastrointestinal tract: one from colon and 6 cases with gastric localization. The aim of the study was to identify the immunoprofile of the tumor cells, as a possible therapeutic target of this rare histologic variant of gastrointestinal carcinoma.
To have a complex immunohistochemical (IHC) picture of these tumors, we assessed the expression of a panel of 39 biomarkers that includes markers for diagnosis, epithelialmesenchymal transition (EMT), adhesion molecules, markers of angiogenesis, and predictive markers. The obtained data were correlated with those obtained after a complex review of literature.

Case Selection.
We have retrospectively evaluated seven consecutive cases of primary gastrointestinal PCs (one of the colon and 6 of the stomach), diagnosed by our team in the last four years. No synchronous urothelial carcinoma or lobular carcinoma of the breast was associated with any of the included cases. No preoperative therapy was done.
The signed informed consent was obtained from all of the patients for publication of clinicopathological data.
We reviewed the Hematoxylin and Eosin-(HE-) stained slides to confirm diagnosis and quantify the percentage and microscopic subtype of adenocarcinoma versus carcinoma with plasmacytoid morphology component. All cases presented at least 80% plasmacytoid component (Table 1). It was evaluated based on the presence of round to ovoid discohesive cells, with eccentrically located nuclei ( Figure 1).
The 8th edition of AJCC staging system [8] was used for establishing the pTNM stage. The Dukes-MAC stage was also appreciated based on new literature proposal [9].

Immunohistochemistry.
In all of the cases, a complex immunoprofile of the tumor cells was done to perform differential diagnosis of a primary versus metastatic tumor ( Table 2). The diagnosis of carcinoma with plasmacytoid morphology was suspected in HE and confirmed by double positivity for CD138 and cytokeratins (CK AE1/AE3 and CK7   or CK20) ( Table 3 and Figure 2). As we have mentioned before, at least 80% plasmacytoid component was identified in all of the cases. CD138 marked the plasmacytoid component only, without positivity in the adenocarcinoma/poorly cohesive carcinoma (including signet ring cells) component. No stromal positivity was noted. The EMT was analyzed using the IHC markers of the Wnt pathway E-cadherin, -catenin, N-cadherin, vimentin, and arylsulfatase A and B (ARSA, ARSB). CD44 was used to explore the stemness features of the tumor cells. Those cases showing loss of E-cadherin with membrane to nuclear translocation of -catenin, or negativity for -catenin, were considered as showing EMT. Positivity for N-cadherin and vimentin was also checked for identification of mesenchymal features. As the adhesion molecule V-set and immunoglobulin (VSIG) and SLUG were positive in all of the cases and N-cadherin and smooth muscle actin (SMA) was negative, the tumors were classified as epithelial-type carcinomas (positive for E-cadherin, with membrane expression ofcatenin and negativity for vimentin) or mesenchymal-type carcinomas (negative for E-cadherin, with nuclear expression of -catenin or positivity for vimentin). The other cases were considered as having a hybrid EMT phenotype (Table 3 and Figure 3).

Clinicopathological Data.
In our university hospital, there are about 60 gastric carcinomas and 150 colorectal carcinomas diagnosed every year. The 7 primary carcinomas with plasmacytoid morphology (one from upper rectum and 6 from stomach) represented about 0.16% of all colorectal carcinomas and 2.5% of all gastric carcinomas diagnosed in our department of pathology during 2016-2019. They were identified in patients with a median age of 70.43±11.24 years (range: 52 to 83 years) and a report of M:F=2.5:1.
All of the patients were diagnosed in metastatic stages, with invasion in lymphatic (L1) and/or blood vessels (V1) and extremely short overall survival ( Table 1). All of the tumors were removed with free resection margins (R0). Only one patient (the youngest one: 52 years old) is alive at 25 weeks after surgery (case 3). As the tumor cells did not express HER-2, classic chemotherapy was administrated. The other 6 patients died between 3 and 23 weeks (below six months) after surgery (Table 1).

Histological Diagnosis.
The diagnosis of a primary carcinoma was histologically based on the origin of tumor cells within gastric or colorectal mucosa. In carcinoma with plasmacytoid morphology of the upper rectum (Case 1, Table 1), it was about a mucinous adenocarcinoma with signet ring cell component, with plasma cells-like discohesive cells, in the invasion front ( Figure 1). As the surgical intervention was made in emergency, due to mechanical ileus, no preoperative chemoradiotherapy was done.
All of the six gastric carcinomas were of poorly cohesivetype, with/without signet ring cell component, with 10% component of adenocarcinoma, in cases 2 and 6 ( Table 1).

Primary versus Metastatic
Tumor. The colonic origin was proved by positivity of tumor cells for CK20 and inconstant positivity for CDX2. Gastric origin was revealed by inconstant positivity for CK7 and/or CK20 (Figure 2). Lymphoma was excluded based on negativity for Leukocyte Common Antigen (LCA), CD20, and CD3, and three neuroendocrine markers (chromogranin, synaptophysin, neuron specific enolase [NSE]) were used to exclude a neuroendocrine carcinoma (Table 3).  Metastases from a PUC were excluded based on positivity for CK20 and/or CDX2 and carcinoembryonic antigen (CEA) and negativity for GATA3. Metastases from a lobular carcinoma of the breast were based on negativity for Estrogen and Progesteron receptors (ER, PgR) and also negativity for mammaglobin, endothelial transcription factor 3 (GATA 3), and gross cystic disease fluid protein 15 (GCDFP-15). S100 and HMB45 negativity excluded a metastatic melanoma (Tables 3  and 4).

Epithelial-Mesenchymal
Transition. From 6 gastric carcinomas with plasmacytoid morphology, 4 cases were classified as mesenchymal-type carcinomas, one as epithelial type, and one as having a hybrid phenotype (positivity for VSIG and SLUG only), the same as the mesenchymaltype carcinoma with plasmacytoid morphology of the upper rectum ( Table 3). The longer survival (over 25 weeks) was seen for the epithelial-type carcinoma (case 2) (Tables 1 and 3 and Figure 3).

Angiogenesis.
All of the cases showed negativity for Vascular Endothelial Growth Factor A (VEGF-A), as expression of lack of angiogenic immunophenotype. Negativity for maspin was an indicator of high risk for distant metastases.

Predictive Markers.
No positivity for HER-2, c-KIT, NGAL, or CD10 was observed but all of the cases diffusely expressed c-MET protein (Figure 4).

Discussion
To differentiate a primary carcinoma with plasmacytoid morphology of gastrointestinal tract from a metastatic tumor, especially from PUC or breast lobular carcinoma, a complex immunoprofile is necessary (Table 4). Primary lymphomas, as plasma cell lymphoma, and carcinoma variants should also be excluded [3].
CK AE1/AE3 marks 97% of carcinomas with plasmacytoid morphology [3], independently of their localization.  Regarding the CK variants, CK20 is usually positive for colorectal carcinomas but gastric carcinomas with plasmacytoid morphology and PUCs can also express this marker. The gastrointestinal versus urothelial origin cannot be based on keratin 7. It is positive for urothelial carcinoma but can also mark the gastric and colorectal carcinomas, especially those with microsatellite status or with serrated pathway and BRAF mutations [7,10]. CDX2 and polyclonal carcinoembryonic antigen (p-CEA) may mark both colorectal carcinomas with plasmacytoid morphology and PUCs but uroplakin is positive for PUCs only [1,4]. CDX2 is rarely positive in gastric carcinomas, as in our cases.
The suspicion of a metastasis from a lobular carcinoma of the breast is eliminated based on negativity for specific markers such mammaglobin, ER, PR, and GATA 3 [1,3,11,12]. However, the poorly cohesive gastric carcinomas can express ER [1,11,12] and urothelial carcinoma can be diffusely positive for GCDFP-15 [1] and express nuclear GATA3 [1,3]. CD138 can mark the breast lobular carcinoma but its expression is simultaneously seen in tumor and stroma cells [13]. E-cadherin is negative in over 75% of invasive lobular carcinoma of the breast, as a result of mutations of the CDH1 gene [13,14]. Loss of E-cadherin occurs in parallel with decreased -catenin expression [14]. GCDFP-15 is commonly negative in invasive breast lobular carcinoma. Whereas HER-2, ER and PR are used as predictive factors, c-MET aberrations (mutations or amplification) are indicators of high-grade invasive breast lobular carcinomas with increased metastatic risk and are commonly identified in triple negative basal-like cases [15] that represent below 2% of all invasive lobular carcinomas of the breast [16].
Similar to our study, it was shown that E-cadherin is mostly negative in PUC, as marker of aggressivity and activated Wnt pathway [2,5,6] but vimentin can be positive or negative [4]. S100, a marker of EMT, was also found negative in the reported PUCs [6], as in our cases.
PUC shows a predilection for intraperitoneal spread and carcinomatous ascites [3,4]. As CA-125, the marker usually used for diagnosis of ovarian cancer, can rise in the serum of patients [3], the differential diagnosis of metastatic carcinoma with plasmacytoid morphology is extremely difficult in females. Similar to colorectal carcinomas, serum CEA can also be high in patients with PUC [4]. CA19-9 and -HCG were also reported to be increased [6]. For any patient with peritoneal carcinomatosis, the primary tumor should be checked in ovary, gastrointestinal tract, and urinary bladder.
As we have mentioned in the Introduction, only one case was reported in literature as gastric carcinoma with plasmacytoid morphology, in a 66-year-old male [4]. Differentiation between a poorly cohesive gastric carcinoma and the carcinoma with plasmacytoid morphology variant is based on CD138 positivity in over 50% of tumor cells [3]. In the present material, the median age of patients was 70.43±11.24 years (range: 52 to 83 years), which is significantly higher than that in other gastric carcinomas previously reported in our department: 62.19±13.96 (range 21-98 years) [9]. The carcinoma with plasmacytoid morphology of the stomach presented, in our material, several negative prognostic factors [8,9,15]: age over 50, advanced stage (both pTNM and Dukes-MAC like), angiolymphatic invasion, EMT phenotype, and positivity for c-MET and CD44. CD44 is a cancer stem marker that seems to induce chemoresistance [12].
As CD138 is an extracellular matrix receptor involved in intercellular communication, proliferation, angiogenesis, and metastasis [3], we consider that it should be considered as an indicator of poorly cohesive carcinoma aggressivity, independent of the tumor location. It probably interacts with the Wnt and ARSA/ARSB pathways and is involved in the process of EMT of carcinoma cells.
Our case series showed that, in gastrointestinal tract, carcinoma with plasmacytoid morphology is an aggressive "mesenchymal-type poorly cohesive carcinoma" that expresses c-MET but not HER-2. This immunophenotype indicates a possibility of the response of these tumors to tyrosine kinase inhibitors that target MET signaling, such as imatinib or foretinib, which are currently used in clinical trials, in patients with solid tumors. This aspect should be proved in large cohorts.

Data Availability
The clinicopathological data used to support the findings of this study are available from the corresponding author upon request.