Carcinosarcoma of the Gallbladder with Chondrosarcomatous Differentiation and Intracytoplasmic Eosinophilic Hyaline Globules (Thanatosomes): A Report of a Case and Review of the Literature

A 52-year-old woman presented with abdominal pain and vomiting. Computed tomography (CT) scan of the abdomen revealed a huge exophytic gallbladder mass displacing or invading the surrounding structures. The patient underwent radical cholecystectomy, transverse colectomy, distal gastrectomy, and liver bed resection. Histologically, the tumor showed both carcinomatous and sarcomatous components, with prominent chondrosarcomatous differentiation. In addition, several malignant cells showed intracytoplasmic eosinophilic hyaline globules (Thanatosomes). The tumor showed metastatic deposits to the omentum, the liver, and the peripancreatic lymph nodes. We report this unusual case and present a review of all cases of carcinosarcoma of the gallbladder with chondrosarcomatous differentiation.


Introduction
Carcinosarcoma of the gallbladder, also referred to by some authors as malignant mixed tumors and sarcomatoid carcinoma, is a rare malignant neoplasm representing less than 1% of all gallbladder malignant tumors [1]. This highly malignant tumor is composed of a carcinomatous component, which can be glandular or squamous or both, intermingled with sarcomatous elements which can include undifferentiated spindly homologous component, or heterologous component including osteosarcomatous, chondrosarcomatous, or rhabdomyosarcomatous differentiation. The presence of heterologous elements differentiates carcinosarcomas from spindle and giant cell undifferentiated carcinomas [1]. These tumors are highly aggressive and behave like a carcinoma. In a review of 3038 patients with gallbladder cancer collected by the Surveillance, Epidemiology, and End Results (SEER) Program, 11 patients with carcinosarcoma were identified and 9 of them died as a result of the tumor [2]. examination, there was a palpable abdominal mass in the right upper quadrant that is 8 cm below the costal margin.
Abdominal ultrasound showed the gallbladder to have thick wall with large irregular shaped soft tissue mass arising from the fundus, measuring 8 x 6 cm. This was seen infiltrating the surrounding sub-hepatic fat planes and inseparable from the transverse colon. There were no focal hepatic lesions; dilated intrahepatic biliary radicals or significant enlarged lymph nodes were noted. The patient was referred to our hospital for evaluation and management. Laboratory work-up revealed normal hematological parameters. Liver function tests revealed an elevated GGT (65 unit/liter), low albumin (32 g/L) and unremarkable total bilirubin, alkaline phosphatase, ALT, and AST. Renal function tests were within normal limits. Serum carcinoembryonic antigen (CEA) and -fetoprotein (AFP) were normal, but carbohydrate antigen 19-9 (CA 19-9) was elevated (154.33 IU/ml). CT scan of the abdomen showed a gallbladder mass with huge exophytic component, displacing the surrounding structures with few small portal lymph nodes ( Figure 1). No hepatic lesions were identified and a few small bilateral pulmonary nodules of uncertain significance were noted. The patient underwent laparotomy with radical cholecystectomy, transverse colectomy, distal gastrectomy, omentectomy, and liver bed resection.

Pathological Findings
The gross pathological specimen consisted of portion of liver with attached gallbladder, portion of stomach, segment of large bowel and attached omentum. The gallbladder measured 13.0 cm in length and 6.0 cm in diameter. On opening the gallbladder, a large polypoid tumor mass was seen filling the lumen measuring 11.0 cm in length and 6.0 cm in diameter. The tip of the mass was friable reddish brown in color. The root which was mainly present in the body is soft reddish tan in color. No gallstones were identified in the lumen of the gallbladder. On cut section, fleshy tumor was seen arising within the gallbladder extending to surrounding tissue, forming rubbery soft tan and hemorrhagic nodules the largest measured 11.0 cm in diameter, reaching to the wall of the stomach and colon surrounded by omental tissue. A large nodule measuring 8.0 cm beneath the omentum adherent to the liver was noted. Another hemorrhagic nodule measuring 7.8 cm was present between the liver and the stomach. On opening the stomach and the large bowel, the mucosal linings of both were unremarkable.
Histological examination of the intraluminal large polyp of the gallbladder showed a biphasic highly malignant tumor featuring both solid epithelial nests within highly malignant sarcomatous stroma (Figures 2(a) and 2(b)) with scattered islands of malignant cartilage seen (Figure 2(c)). The tumor cells had hyperchromatic pleomorphic nuclei with many huge monstrous bizarre multilobulated forms noted (Figure 2(d)). Many mitoses, mostly atypical (tripolar), were seen. In addition, several scattered tumor cells showed the presence of intracytoplasmic eosinophilic hyaline globules (EHGs) that were variable in size from finely small granules to large globules (Figures 2(e) and 2(f)). Some EHGs were seen in adjacent connective tissue stroma to disrupted or degenerated cells that contained these globules. The wall was heavily infiltrated by the tumor in the area that the polyp had arisen from. Sections from the gallbladder away from the tumor showed evidence of xanthogranulomatous cholecystitis.
Infiltrating highly sarcomatous component was seen in the pericolic fat, perigastric fat, hepatic parenchyma, and perihepatic fibrous tissue. Foci of malignant solid epithelial nests and malignant glandular formation surrounded by sarcomatous stroma with scattered islands of malignant cartilage Case Reports in Pathology In view of these findings, the diagnosis of gallbladder carcinosarcoma with anaplastic sarcomatous and chondrosarcomatous components was confirmed. This neoplasm was staged as stage III (pT4 pN2 pMx).
The postoperative course was uneventful. The patient was referred to the Medical Oncology team 7 weeks following the surgical procedure. A management plan was set and included six cycles of adjuvant chemotherapy (gemcitabine 1000 mg/m 2 and cisplatin 25 mg/m 2 ). Upon the patient's request, she continued her chemotherapy treatment near her home and no further follow-up information was obtained afterwards. Their search included cases labelled as carcinosarcoma, sarcomatoid carcinoma, and spindle cell carcinoma of the gallbladder [4]. They selected only cases with a reliable histopathological diagnosis of CSGB. Since then, additional several cases had been reported.

Discussion
There had been some controversy regarding the pathogenesis of this tumor, reflected by the multitude of names designated for it, including malignant mixed tumor [5][6][7], sarcomatoid carcinoma [8][9][10][11], spindle cell carcinoma [12,13], and malignant mixed mesodermal tumor [14]. Five theories were postulated to explain the histological mixture of epithelial and mesenchymal malignant elements in CSGB. One theory proposed that the mesenchymal tissue represented a reactive process; a second theory considered the mesenchymal tissue as a true sarcoma (the collision tumor or convergence multiclonal theory); a third theory hypothesized that the tumor is a carcinoma with metaplastic changes to sarcoma (the divergent monoclonal theory); a fourth theory pointed to embryonic rest origins; and the last theory was the totipotential stem cell hypothesis [15,16].
To add to this controversy regarding its pathogenesis, CSGB had been categorized as a "true" carcinosarcoma if the mesenchymal elements showed differentiation into neoplastic bone, cartilage or skeletal muscle [1,40], or as "so-called" carcinosarcoma if the malignant mesenchymal component did not show differentiation, but exhibited spindly or pleomorphic cells without differentiation into malignant bone or cartilage or rhabdomyoblastic cells [8,12]. Adenocarcinoma is almost always a constant finding in the carcinomatous component of CSGB, but in one third there are foci of keratinizing squamous cell carcinoma [1].  [16]. A mixture of more than one heterologous element such as malignant osteoid and cartilage were observed in several cases [4]. Zhang et al. divided 45 cases of CSGB they retrieved from the literature that had details of their histological findings. They divided the cases into five groups by their mesenchymal component type, including spindle cell, chondroid, rhabdomyomatous, osteoid, and admixture of two or more component types. They found on statistical analysis no significant difference in the outcome of patients with various histological types, thus suggesting that mesenchymal component of CSGB has little prognostic value [4].
We collected all cases of CSGB that exhibited chondrosarcomatous differentiation (Table 1). There were 30 cases, including the current one. The cases were collected from the world literature over 93 years dating back to 1925 [4-7, 13, 17-39]. There were 8 males and 21 females, with one case without available data on age and sex of the patient [19]. Male to female ratio was 1:2.6. Their ages ranged from 38 to 91 years (Mean age 67 years). In addition to the presence of chondroid differentiation, twelve tumors showed additional osteoid differentiation [5,7,17,21,22,25,28,31,33,36,38,39], two cases showed additional rhabdomyosarcomatous differentiation only [7,26], and one case showed additional both rhabdomyosarcomatous and osteoid differentiation [24]. Metastatic spread, particularly Case Reports in Pathology 5  Developed leakage at anastomosis site and severe infection. Developed many liver metastases, and died of liver failure on 61st post-operative day.  to the liver was documented in 18 cases (62%). Data related to survival, excluding autopsy cases, were available on 20 patients. Two patients died within days following surgery due to massive pulmonary embolism [7], or cardiogenic shock [32]. Twelve patients died of metastatic spread of the tumor or sepsis within a period of time ranging from 1 month to 17 months (mean 4.9 months), six of them within three months. Six patients were alive at the time of last follow up, with their survival ranging from 3 to 54 months (mean 33.8 months). Allover survival was 13.5 months, ranging from 0 to 54 months. Prolonged survival had been emphasized in two patients, one survived 48 months [31], and the other 54 months [29].
In a review of 59 cases of CSGB that underwent surgical resection, there were 18 males and 39 females, with male to female ratio of 1:2.2. Two-thirds of the patients (38 patients) were 65 years or older. The survival rate by the end of one year was 42.8%, and the 5-year survival rate was 37.6%. The median survival in months was 7.0 ± 2.0 [3]. Another review of 36 patients of CSGB, who underwent surgical management, showed median survival to be 7.0 months with a range from 4.4 to 9.6 months. There were 10 males and 26 females with male to female ratio of 1:2.6, with a mean age of 67.7 years [42]. A meta-analysis study of 68 cases of CSGB showed the mean age of the patients was 68.8 years. There were 16 males and 52 females with male to female ratio of 1:3.25. The mean survival was 17.5 months, with a median of 5 months, and a range between 0 and 85 months [4].
Comparing the data from these studies to ours, the dominance of CSGB in the females is apparent. The mean age is very close in all four studies, ranging from 65 to 68.8 years. Survival data were similar There are some variations in the mean of survival in months following surgical intervention, but is not greatly significant. One of the interesting points made in the meta-analysis study was correlation of the race of the patients with survival. They found that Japanese patients (27 cases) had longer survival time than non-Japanese (24 cases), with mean survival of 19.9 months for the Japanese vs 11.5 months for the non-Japanese, with a median of 6 vs 4 months. In our review, 11 out of 29 patients (38%) were Japanese, with average survival of 16.9 months, compared with nine non-Japanese, with available data on their survival, who had average survival of 6.2 months. Three of the Japanese patients had prolonged survival of 24, 48, and 54 months, respectively (Table 1). Japanese patients with carcinoma of the gallbladder had better survival when compared with other high incidence countries such as Chile and India. Multiple factors might result in such better prognosis in Japanese, including genetic variations, early detection, and better surgical techniques [4].
Abdominal mass, fever, nausea, vomiting, abdominal pain, anorexia, loss of weight, and painless jaundice are some of the nonspecific presentations of this tumor [24,31]. Our patient was admitted initially to the Emergency Department due to abdominal pain with vomiting but without jaundice. An abdominal mass was palpated and was confirmed by ultrasound examination. CSGB are usually polypoid and their size varied from 3 to 15 cm. and can fill the lumen of the gallbladder [1]. Our patient had a tumor that filled the lumen measuring 11 cm in maximum dimension. In a review of 41 patients with CSGB who had data on tumor size and survival, it was demonstrated that patients with tumors measuring ≥ 5 cm had a significantly shorter survival when compared with patients who had tumors measuring less than 5 cm. [4].
Gallstones were observed in 8 out of 9 cases (88%) of CSGB in one review [24], in 14 out of 18 cases (77%) in another review [23], and in 66.7% of cases in a third review [4]. This high association between gallstones and CSGB is similar to what is observed with carcinoma of the gallbladder. Our patient did not have gallstones found in the lumen of the gallbladder but got xanthogranulomatous cholecystitis.
Historically, the concept of "carcinosarcoma" was introduced by Rudolf Virchow in 1865 [43]. Karl Landsteiner, an Austrian pathologist and the discoverer of the ABO blood groups, was credited with the description of the first case of CSGB in 1907 in a pathology museum specimen [44]. Saphir and Vass made in 1938 a review of all cases of carcinosarcoma in various organs, dating back to 1878 [45]. They collected 153 cases from 25 organs, with most frequent cases encountered in uterus, breast, lung, larynx, esophagus, thyroid, and urinary bladder. There were only two cases of CSGB, including Landsteiner's case. They were of the opinion that majority of these tumors are of carcinomatous origin, and their critical review of the documented cases indicated that their carcinosarcomatous nature was questionable. It is interesting to note that these authors made their observations on routinely H&E stained histological sections fifty years before the introduction of immunohistochemical stains as a diagnostic tool that demonstrated the positive staining of the spindly "sarcomatous" for cytokeratin and other epithelial markers confirming their nature as epithelial in origin.
To the best of our knowledge, none of the cases of CSGB on record had demonstrated the presence of eosinophilic hyaline globules (EHG). The present case showed the presence of several EHG within the cytoplasm of the mesenchymal sarcomatous spindly and pleomorphic cells, as well as free within the surrounding extracellular tissue. They were variable in size and were highlighted by Periodic Acid Schiff (PAS) stain. Such EHG had been labelled as "Thanatosomes," a term proposed by Papadimitriou et al. denoting a degenerative phenomenon related to apoptosis, common to all cell types, benign or malignant, resulting from cytoplasmic blebbing and nuclear fragmentation followed by phagocytosis by neighboring cells or auto-phagocytosis, accompanied by increased membrane permeability to plasma proteins [46]. In their study, they examined 80 tumors from various organs that contained hyaline globules by light and electron microscopy and did immunohistochemical studies on them. Most cells containing EHG stained with immunohistochemical stains for all plasma proteins examined alpha-1-antitrypsin, ferritin, C3, kappa and lambda light chains, and IgG, indicating increased plasma membrane permeability. Among those 80 tumors, there were two cases of hepatocellular carcinoma but no cases of gallbladder cancer.
In another study related to Thanatosomes in gastrointestinal epithelium, Dikov et al. examined 2230 biopsies of normal and pathological epithelium. EHGs were very rarely found in normal epithelium (1.1%), but their number increased significantly in specimens with ischemic injury (47%) and benign regenerative proliferation (70%). They concluded that Thanatosomes represented a relatively constant and useful histologic marker of enhanced cell turnover and ischemic injury [47]. Thanatosomes had been dubbed as "Death Bodies" in a report on 2 cases of malignant phylloides tumor, both of which showed numerous Thanatosomes, to the point of dominating the histological appearance and masking the stromal element [48]. Our case is the first CSGB to feature this peculiar finding.

Disclosure
This work was presented as a poster presentation at the 31 st Congress of the International Academy of Pathology (IAP) held in Cologne, Germany, on 25-29 September 2016.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this article.