Case report
The patient was a 56-year-old man who was followed up for alcoholic hepatitis and cirrhosis for 4 years. His medical history included obesity, diabetes mellitus and Crohn’s disease, which had been continuing for 7 years. He was admitted to our emergency clinic with amnesia, debility, apathy and aphasia. Physical examination revealed a mild abdominal distension related to ascites. Imaging findings of the liver (ultrasonography, doppler, and computed tomography indicated irregular contours and diffuse granular heterogeneity of the parenchyma (Fig. 1). Laboratory tests demonstrated hypoalbuminemia (0.49 g/dL), hyperbilirubinemia (total bilirubin: 2.55 mg/dL and direct bilirubin: 1.80 mg/dL) and hyperammonemia (197.61 μg/dL). Tests for viral and autoimmune markers were negative. Aspartate aminotransferase (30 U/L), alanine aminotransferase (45 U/L) and alkaline phosphatase (65 U/L) were in normal ranges. The serum alpha-fetoprotein (AFP) level was 2.8 ng/mL. Other tumor markers, such as carcinoembryonic antigen and carbohydrate antigen 19-9 were also within the normal range.
Because of the rapid progress of the liver failure, a liver transplantation was performed. Serum albumin (0.60 g/dL), aspartate aminotransferase (26 U/L), alanine aminotransferase (13 U/L) and alkaline phosphatase (73 U/L) were tested preoperatively. There was no evidence of hepatic failure, recurrence or metastasis in the 10 months following surgery. Moreover, no serological endocrine markers were detected (i.e. chromogranin A, gastrin, glucagon and vasoactive intestinal polypeptide) after the pathological diagnosis.
Methods
The liver specimen was fixed in 10% buffered formalin, embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin. Immunohistochemistry was performed on deparaffinized tissue sections using the avidin-biotin-peroxidase method and a Dako AutoStainer (Carpinteria, CA, USA). After antigen retrieval, the following panel of primary antibodies was applied: Hep Par1 (hepatocellular antigen, 1:50; Dako), CD34 (1:200; Dako), synaptophysin (1:10; Dako), chromogranin (1:50; Dako), CK7 (1:1,500; Dako), CK19 (1:50; Dako), CD56 (1:50; Vision BioSystem), MIB-1 (1:100; Dako) and β-Catenin (1:250; Dako).
Gross findings
The resected specimen consisted of a 18×14×9 cm hepatectomy with an attached gallbladder, consisting of 800 g. The external surface was coarsely nodular. Cut sections showed well-demarcated nodules, measuring from less than 1 cm to up 3 cm in greatest dimensions. Between these nodules, a well circumscribed and demarcated but non-encapsulated region of masses was present. The region was composed of two sharply defined and distinctive areas, including a green area with 1.7×1×1.6 cm dimension and the second being a distinctly yellow-tan colored area with lesions having the largest dimension of 0.6 cm (Fig. 2). No satellite lesions were identified. The gallbladder was normal.
Histopathological findings and immunohistochemistry
The green and nodular component consisted of tumor cells that were arranged in cords and plates of various thicknesses that were separated by sinusoid-like areas lined by flat CD34-positive endothelial cells. Tumor cells were large with prominent nucleoli and abundant eosinophilic cytoplasm and showed a strong positive staining with Hep Par-1. Immunohistochemistry staining for synaptophysin, chromogranin, CD56, CK7 and CK19 was negative (Fig. 3). These findings corresponded to a moderately differentiated HCC.
The second smaller distinct component of the tumor was formed from small uniform tumor cells with a narrow cytoplasm and condensed chromatin, giving the nuclei a hyperchromatic appearance. These cells were arranged in a trabecular fashion and as nests. The presence of tumor cells in the vicinity of the ductal structures was also observed. Atypical mitosis was 5/10 in high power field. Immunohistochemistry staining showed positivity for chromogranin, synaptophysin, NSE, CD56 and CK7, but negativity for Hep Par1 and CK19. For MIB-1, immunostaining of the endocrine component indicated a proliferative activity of 18%. These findings were in accordance with NEC grade 2 (G2) (Fig. 3). A broad fibrous band without a transition area separated the tumors from each other. While a strong β-catenin membrane staining was observed in the HCC area, the NEC area did not show such staining (Fig. 3).
Discussion
The case presented here was diagnosed as collision tumor type of HCC-NEC, according to the pathological findings. Although two tumors were observed concurrently, a transition between them was not observed.6–10 These findings are in contrast with combined tumors that consist of two distinct tumors that are contiguous and intermingle with each other.11–19,21 Another important problem to be solved in making a diagnosis of primary hepatic NEC is the discrimination of primary NEC and from metastatic NEC, as the liver is the most frequent site of metastasis for NEC.22 Therefore other primary sites should be examined when an NEC is suspected in the liver.14 In our case, there were no other neoplastic lesions found besides in the liver.
The clinical and pathologic findings of the cases described in the literature are presented in Tables 1 and 2. The present case involved a 56-year-old man who had undergone liver transplantation because of chronic liver failure. The diagnosis of a collision type of HCC-NEC was made after the pathological examination of the resection specimen. The age and sex of the patient are in agreement with previously reported cases (mean: 64.4 years; male/female: 20/1) and support that these tumors are more frequently seen in older age groups and men.
Table 1.Demographic, laboratory, clinical and macroscopic findings of previously reported HCC-NEC cases
| Reference, year | Age | Sex | Symptoms | Virus | AFP | Diagnosis | Metastasis | D, in cm |
| Barsky et al.11, 1984 | 43 | M | Abdominal swelling | HBV | ↑ | Autopsy | Omentum | L |
| Artopoulos et al.12, 1994 | 69 | M | Abdominal pain | HBV | ↑ | FNA | – | 10 |
| Vora et al.13, 2000 | 63 | M | Abdominal pain, jaundice | NA | NA | NA | NA | 10 |
| Ishida et al.6, 2003 | 72 | M | – | HCV | ↑ | Resection, LND | LN | 3 |
| Yamaguchi et al.14, 2004 | 71 | M | – | HCV | ↑ | Resection | – | 4.1 |
| Garcia et al.7, 2006 | 50 | M | – | HCV | ↑ | Resection | – | 5.3 |
| Yang et al.15, 2009 | 65 | M | Epigastric pain | HBV | → | Resection, LND | LN | 7.5 |
| Tazi et al.8, 2011 | 68 | M | – | HBV | ↑ | Resection | – | 4.0 |
| Nakanishi et al.16, 2012 | 76 | M | – | HCV | ↑ | Resection | – | 3.0 |
| Hammedi et al.17, 2012 | 51 | M | Abdominal pain | HCV | ↑ | Resection | – | 20 |
| Aboelenen et al.18, 2014 | 56 | F | Abdominal distension | – | ↑ | Biopsy | LN | NA |
| Nishino et al.19, 2016 | 72 | M | – | HCV | ↑ | Resection | – | 2.5 |
| Choi et al.9, 2016 | 72 | M | – | HCV | → | Resection | – | 2.5 |
| Nomura et al.5, 2016 | 71 | M | NA | HCV | ↑ | Resection | NA | 4 |
| 71 | M | NA | HCV | → | Resection | NA | 3 |
| 58 | M | NA | HBV | ↑ | Resection | NA | 4.3 |
| 50 | M | NA | HBV | ↑ | Resection | NA | 1.8 |
| 63 | M | NA | HCV | ↑ | Resection | NA | 3 |
| Baker et al.21, 2016 | 76 | M | NA | – | ↑ | Resection | – | 5.5 |
| Liu et al.10, 2016 | 65 | M | Abdominal discomfort | HCV | ↑ | Resection | LN | 4.3 |
| Okumura et al.20, 2017 | 70 | M | Solid mass | HCV | → | Resection | – | 11 |
| Present case | 56 | M | Incidental | – | → | Resection | – | 2.3 |
Table 2.Pathological and clinical findings of previously reported HCC-NEC cases
| Reference, year | N | C | Type | Therapy | Recurrence | Time* | ST |
| Barsky et al.11, 1984 | S | + | Combined | Adriamycin, 5-FU | Unresectable | 26 | D |
| Artopoulos et al.12, 1994 | S | + | Combined | – | NA | NA | NA |
| Vora et al.13, 2000 | S | + | Combined | – | NA | 1 | D |
| Ishida et al.6, 2003 | M | + | Collision | – | NA | NA | NA |
| Yamaguchi et al.14, 2004 | M | – | Combined | – | Pelvic bone | 5 | A |
| Garcia et al.7, 2006 | S | – | Collision | TACE, doxorubicin | Liver, peritoneum | 16 | A |
| Yang et al.15, 2009 | S | – | Combined | – | Liver, adrenals, paraaortic LNs | 12 | D |
| Tazi et al.8, 2011 | S | – | Collision | Cisplatin, etoposide | – | 28 | A |
| Nakanishi et al.16, 2012 | S | NA | Combined | TACE, epirubicin lipiodol | Sacral bone | 17 | D |
| Hammedi et al.17, 2012 | S | – | Combined | – | – | 6 | A |
| Aboelenen et al.18, 2014 | S | – | Combined | – | – | 1 | D |
| Nishino et al.19, 2016 | | – | Combined | Cisplatin, etoposide | Regional, paraaortic LNs | 2 | D |
| Choi et al.9, 2016 | M | – | Collision | Cisplatin, etoposide | Liver | 10 | A |
| Nomura et al.5, 2016 | S | – | Combined | – | Liver | 8.6 | D |
| S | – | Collision | – | Diaphragm, liver | 2.6 | D |
| S | – | Combined | – | – | 19.7 | A |
| S | + | Combined | – | – | 19.5 | A |
| S | – | Combined | | – | 24 | A |
| Baker et al.21, 2016 | S | + | Collision | Cisplatin | – | NA | A |
| Liu et al.10, 2016 | S | + | Collision | – | NA | 1.3 | D |
| Okumura et al.20, 2017 | S | – | Collision
Combined | – | LNs | 3 | D |
| Present case | S | + | Collision | – | – | 10 | A |
Because many patients are followed up for chronic viral hepatitis (hepatitis B virus, 30%; hepatitis C virus, 60%), tumor-related symptoms are absent in most cases (50%). In symptomatic cases, abdominal pain is the most common finding (Table 1). Our patient was being followed up for cirrhosis related to alcoholic hepatitis and did not have any tumor-related symptoms. Laboratory investigation of tumor markers in recently reported cases demonstrated that AFP was frequently high in 76% of the cases. On the other hand, similar to the case presented here, AFP was within normal ranges in 19% of cases. Other tumor markers, such as carcinoembryonic antigen,7,14–16,18,19 carbohydrate antigen-19-97,8,15,16,18,19 and carbohydrate antigen-1257,18 were not found to be predictive in the diagnosis of these tumors. PIVKA-II (protein induced by vitamin K deficiency or antagonists-II) level was normal in a case reported by Nakanishi et al.16 Only one case displayed a high level of PIVKA-II.19
These data indicate that although these markers are valuable in the discrimination of malignant tumors, they cannot completely rule out some malignancies, such as combined or collision type of HCC-NEC, especially in which cases the AFP level might be normal. Radiological findings of these lesions are not specific and their discrimination is not always possible. Parallel to these observations, in our case neither the tumor nor its nature could be clarified by the radiological investigation. In 80% of the recently reported cases, the diagnosis necessitated surgical resection. Only 2 cases were diagnosed by fine needle aspiration and liver biopsy and 1 case was diagnosed at autopsy (Table 1). Therefore, the diagnosis of these tumors relies on a comprehensive macroscopic and microscopic evaluation together with additional methods, including immunohistochemistry.
Although multiple HCC-NEC of the liver has been described in some reports,6,9,14 they are solitary lesions and frequently do not metastasize at the time of diagnosis, as seen in our case. Rarely, metastases to lymph nodes and omentum have been described (Table 2).6,10,11,15,18 An important problem in these cases is the difficulty to detect which component of the tumor determines the behavior of the tumor. In a great majority of recent cases, only the NEC component has been detected in the metastatic focus.7,8,14–16,19 This observation suggested that NEC components strongly affect the aggressive behavior of the tumor and it is proposed that the NEC component may be responsible for the poor prognosis in these patients.16,19 Recently, Choi et al.9 proposed adjuvant chemotherapy to increase life expectancy when the grade of NEC is high. Because the awareness of an NEC component might influence the treatment strategy and eventually the outcome of the patient,9,16,19 the possibility of NEC should be considered in HCCs with nodal metastasis.
According to the literature, the mean diameter of HCC-NEC is 6.23 cm (Table 1). The mean diameter of the tumor presented here was 2.3 cm. When combined type and collision type HCC-NEC are analyzed separately, the mean diameter of combined type tumors (6.2 cm) is larger than that of collision tumors (3.94 cm). However, with the few cases reported it is not possible to make a definitive conclusion about this finding. On the other hand, this data indicates that a careful macroscopic examination has paramount importance in the detection of HCC-NEC in resection specimens from patients who suffer from chronic liver diseases.
A significant number of reported HCC-NEC cases (62%) have developed in noncirrhotic livers (Table 2). In our case, the presence of the tumor in a cirrhotic background is in contrast to this finding. However, 3 of the 7 reported collision tumors of HCC-NEC were also detected in cirrhotic background.6,10,20 These data support that HCC-NEC should be considered either clinically and pathologically in all patients with chronic liver diseases, regardless of the presence of cirrhosis.
In the present case, no recurrence was observed during the follow-up (10 months) in accordance with the follow-up information for the previously reported cases of HCC-NEC (Table 2). However, 7 of 12 cases of combined tumor of HCC-NEC with follow-up information reportedly died from the disease (Table 2). The mean survival time is 11.9 months for all reported cases (Fig. 4). Although these data point to an aggressive behavior of these tumors, more cases should be reported to delineate the behavioral difference between combined type and collision type of HCC-NEC. Unfortunately, there is no specific treatment protocol due to the small number of cases (Table 2).
Although the origin of primary NEC in the liver is not clarified, two hypotheses have been proposed recently. The first hypothesis is that these tumors originate from hepatic stem cells from intrahepatic bile ducts.23,24 This hypothesis is supported by the occurrence of neuroendocrine tumors in noncirrhotic livers. In a recent report, CK19 expression was observed in the NEC portion of the tumor, supporting the role of hepatic stem cells in the evolution of primary NEC.7 Although in our case CK19 was negative, the localization of tumor cells in the vicinity of ductal structures was an interesting finding. The other hypothesis is neuroendocrine differentiation of stem cell precursors of malignant cells from another liver tumor. Parallel to this opinion, many HCC with neuroendocrine features have been reported.25 Moreover, the expression of neuroendocrine markers in the HCC component of combined HCC-NEC has been observed.6,15 In 1 case, the presence of neurosecretory granules was also detected in the HCC component under electron microscopy.15 Therefore, it is possible to postulate that these combined tumors arise from stem cells that evolve into divergent differentiation. On the other hand, such findings did not describe collision type HCC-NEC tumors. Because our case was incidentally observed and the diagnosis relied on histopathological and immunohistochemical findings we did not have the possibility to perform an electron microscopic or molecular investigation.
Recently, in an elegant study, Baker et al.21 detected a CTNNB1 gene mutation (S33F located in exon 3) in both components of a collision tumor of HCC-NEC and suggested a common cell origin. On the other hand, in a recent report, Liu et al.10 did not find similar results by immunohistochemistry. They observed β-catenin staining in both components of the tumor and stated that these findings did not support exon 3 mutations in the CTNNB1 gene. In our case, similar to the case presented by Liu et al.,10 a strong β-catenin staining was observed in the HCC component. However, we did not observe β-catenin expression in the NEC component, leading us to speculate that in these tumors, divergent underlying molecular pathways might take place that involve the β-catenin pathway. Moreover, a previous case reported by Okumura et al.21 did not only show that the composite and collision type HCC-NEC could be synchronous but also indicated that understanding of the histogenesis of these tumors is very difficult to define.
In conclusion, although collision tumor of HCC-NEC of the liver is very rare, the case presented here supports their presence in this localization in older age groups and in men. Moreover, the present case and the review show that the rare occurrence of HCC-NEC of the liver and lack of diagnostic clinical signs and symptoms do not exclude their consideration in the differential diagnosis of liver tumors, especially in patients with the chronic liver disease.