Two autopsy cases of desmoplastic small round cell tumor

Desmoplastic small round cell tumor (DSRCT) is a rare aggressive malignant tumor. It is a refractory tumor and the median overall survival is very short. We report two autopsy cases of DSRCT, both of which were already advanced and metastasized at the first medical examination. Both cases showed typical DSRCT findings in terms of localization of the lesions, histopathology and genetics, but the rate of disease progression was quite different. Survival after initial symptoms in Case 1 was only 12 months. On the other hand, survival after primary hospitalization in Case 2 was 42 months. The Case 2 patient initially received chemotherapy for advanced pancreatic carcinoma, because a nodule of the pancreatic tail was found on computed tomography (CT) scan. After chemotherapy, tumor regression was observed on CT scan. It is thus implied that adoption of the regimen for pancreatic carcinoma might have been one of reasons of the long survival in Case 2.


INTRODUCTION
Desmoplastic small round cell tumor (DSRCT) is a rare malignant neoplasm that generally occurs in the peritoneal cavity, retroperitoneum and pelvic cavity of young male adults. 1 DSRCT is genetically characterized by the chromosome translocation (11;22)(p13;q12). 2 The fusion protein EWSR1-WT1 resulting from the translocation has oncogenic properties. 3 Typical DSRCT shows sheet-like structures composed of small round cells surrounded by abundant desmoplastic stroma. 4 Patients with DSRCT are generally considered to have a poor prognosis. A clinical report of 20 abdominal DSRCT cases showed that the median overall survival (OS) was 22 months (interquartile range: 12-28 months), and that the 5-year OS rate was 20%. 5 However, cases of long-term survival of metastatic DSRCT have recently been reported. 6 Here we present two autopsy cases of DSRCT, that is, an aggressive case (survival after initial symptoms was only 12 months) and a long-term survival case (survival after primary hospitalization was 42 months). Table 1 shows the clinical summary of both cases including patient characteristics, clinical and imaging findings, initial diagnosis (clinical/histological), treatments, and outcomes. A large mass in the pelvic cavity of the Case 1 patient was demonstrated by computed tomography (CT) taken at the initial hospitalization (Fig. 1a). The Case 2 patient received FOLFIR-INOX (folinic acid, fluorouracil, irinotecan and oxaliplatin) therapy, which is one of the standard therapies for patients with inoperable pancreatic carcinoma but an unusual therapy for a patient with DSRCT, since the clinical diagnosis was advanced pancreatic carcinoma. 7 The therapy was continued for 17 months, because an obvious tumor reduction effect was seen on CT scan 1 month after start of the therapy. The representative comparison is shown in Fig. 3a.

Case 1
During the operation, surgeons noted massive ascites, multiple nodules in the omentum, and numerous foci disseminated in the rectovesical pouch and mesenterium. The largest omental tumor (approx. 20 cm in diameter) fell into the pelvic cavity. Only large omental nodules were resected. Surgical specimens were whitish nodules with hemorrhage, fibrosis and myxomatous degeneration. Histologically, atypical cells with small round nuclei and scanty cytoplasm mainly formed a solid alveolar structure surrounded by an abundant fibrous stroma. Tumor cells made a cord-like structure at infiltrating lesions. Mitotic figures were frequently observed in the tumor. Immunohistochemically, tumor cells were positive for cytokeratin AE1/3, epithelial membrane antigen (EMA), desmin, CD99, vimentin, and neuron specific enolase (NSE). EWSR1 split signal was detected by fluorescence in situ hybridization (FISH) (Fig. 1b), indicating that the EWSR1 gene was on the locus affected by chromosomal translocation. Although the counterpart of gene fusion with EWSR1 remained unclear, the overall results of testing supported the diagnosis of DSRCT. Although various treatments had been tried after the diagnosis, his general condition progressively deteriorated, and he died 1 year after he noticed sensation of residual urine and painful urination.
Autopsy was performed 2 h after his death. Macroscopically, severe emaciation and massive abdominal distension © 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd were seen. Multiple subcutaneous nodules (max. 7 × 4 cm in the left precordium) were found. Severe edema was noted in the left lower extremity. Yellowish serous effusion was collected from the abdominal cavity (5 L), the left pleural cavity (2.5 L), and the right pleural cavity (0.2 L). Numerous disseminated foci were observed in the abdominal cavity ( Fig. 1c). Multiple metastatic lesions were noted in the liver, pancreas, spleen, lungs, skin, bone marrow, kidneys, adrenal glands and lymph nodes (Fig. 1d). A large whitish nodule (6 × 7 × 9 cm) was found in the pelvis (Fig. 1e). The nodule was mainly composed of fibrotic scarring and degenerated tumor cells, probably due to the stereotactic irradiation. The pleural cavities had a few disseminated foci. Microscopically, variably-sized and -shaped, sharply outlined nests of neoplastic cells surrounded by prominently desmoplastic stroma were observed in the foci (Fig. 2a). Central necrosis and cystic degeneration were frequently seen in the tumor. Most of the neoplastic cells had round hyperchromatic nuclei and scant cytoplasm (Fig. 2b). The results of immunostaining with autopsied specimens reproduced those with surgical specimens (Figs. 2c,d). The antibodies used for the immunostaining are listed in Table S1. In addition, tumor cells were positive for FLI-1, LRRC15 and WT1 (antibody against the C-terminus) ( Fig. 2e left panel and Fig. S1c). 9 With another anti-WT1 antibody directed to the N-terminus, some of the tumor cells showed cytoplasmic reactivity ( Fig. 2e right panel). The discrepancy in positivity between the two antibodies against WT1 was consistent with past reports. 10,11 To identify the counterpart of gene fusion with the EWSR1 gene, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed by using total RNA extracted from a snap frozen tissue of one of the metastatic nodules in the liver, and conventional EWSR1-WT1 fusion transcripts (EWSR1 exon 7 to WT1 exon 8) were detected (Fig. 2f). 2,8 Furthermore, since many isoforms, different from case to case, of EWSR1-WT1 fusion transcripts were generated by alternative splicing, we then performed nextgeneration sequencing analysis, identifying that the commonest type of the known isoforms was expressed in Case 1 (data not shown). Severe atelectasis, probably due to the left pleural effusion, was noted in the left lung (lung weight: left, 240 g; right, 305 g). The left renal pelvis was mildly extended (kidney weight: left, 130 g; right, 135 g). Many metastatic nodules in the liver and spleen resulted in severe hepatosplenomegaly (liver weight, 3600 g; spleen weight, 215 g). He died of protein-energy malnutrition (cachexia) due to systemic metastases and dissemination of DSRCT.

Case 2
Hepatic needle biopsy was performed for histological evaluation. Histopathologically, atypical epithelial cells with small round nuclei and scanty cytoplasm mainly formed a solid alveolar structure surrounded by fibrous stroma. Tumor cells showed immunoreactivity for cytokeratin AE1/3, EMA, vimentin and desmin. They were negative for chromogranin A, synaptophysin, and CD56, but weakly positive for NSE. Hepatic metastasis from pancreatic neuroendocrine carcinoma was suggested. Before completion of the FOLFILINOX therapy, FISH analysis revealed EWSR1 split signal in the biopsied tumor tissue (Fig. 3b). The split signal was different from that in Case 1. One to two of 5′ EWSR1 signals (red) and one to five of 3′ EWSR1 signals (green) per cell were seen. No 3′/5′ EWSR1 signal (yellow), which means the EWSR1 gene locus is normal, was observed. Additional immunostaining was performed to examine the expression of WT1 C-terminus, WT1 N-terminus, and LRRC15 ( Fig. S1a, b, and d, respectively). RT-PCR with extracted RNA from the formalin fixed paraffin embedded tissue of the liver biopsy and direct sequencing detected EWSR1-WT1 fusion transcript (EWSR1 exon 7 to WT1 exon 8) (data not shown). The final diagnosis was DSRCT.
One day before death, severe melena and hemorrhagic shock were noted. Rupture of esophageal varices was considered to be the origin of the hemorrhage. The varices had formed secondarily to portal hypertension, probably due to the multiple liver metastases. A biliary stent was implanted because of hepatic dysfunction. He died 42 months after the initial indication of the lesions including pancreaticohepatic nodules.
nodule was located between the parenchyma of the pancreatic tail and the surrounding adipose tissue, where the primary focus was detected by the initial CT scan. Multiple metastatic nodules were found in the liver, spleen, adrenal glands, esophagus, stomach, rectum, lungs, pleura, mediastinum, bone marrow and lymph nodes. Liver metastasis was especially obvious, and the portal vein was constricted by the exclusion of the metastatic nodules at the porta hepatis (Fig. 3d). Cirrhosis was not observed in the hepatic parenchyma. Severe hepatosplenomegaly was noted (liver weight 3400 g, spleen weight 960 g) (Fig. 3e). Varices were formed in the lower esophagus. Hemorrhage was seen in the stomach. There was a large amount of hemorrhagic content in the colon. Histologically, atypical cells with hyperchromatic round nuclei formed a trabecular and solid structure surrounded by abundant fibrous stroma in the nodular lesions including the omentum and pancreatic tail (Fig. 4a-c). In addition to metastasis, extramedullary erythropoiesis was seen in the congestive spleen and liver (Fig.  4d). Mucosal and submucosal veins were severely dilated in the lower esophagus and stomach (Fig. 4e). The bone marrow was hypoplastic due to osteogenic metastatic foci in the lumber vertebrae (Fig. 4f). The patient died of gastrointestinal hemorrhage from the esophageal and gastric varices, which had formed due to portal hypertension. Stenosis of the portal vein by the exclusion of metastatic nodules in the porta hepatis was considered to be the cause of portal hypertension.

DISCUSSION
In this article, we reported two autopsied DSRCT cases diagnosed by detection of the fusion transcript EWSR1-WT1. 8 The presence of the split signal of EWSR1 revealed by FISH analysis was quite a useful clue in the diagnostic process of both cases. Case 2 showed an atypical signal pattern, that is, loss of normal EWSR1 gene locus and amplification of the locus containing 3′ EWSR1. Another atypical pattern has been reported by Starza et al. 12 In any case, both patterns are compatible with the chromosomal status in DSRCT and significance of these atypical patterns is still uncertain.
Desmoplastic small round cell tumor generally occurs in the abdominal and/or pelvic cavity, and subsequently spreads to systemic organs by means of direct invasion, hematogenous and lymphatic metastases, and dissemination. The majority of DSRCT patients tend to present metastasis even at their clinical onset, as was the case with our two cases. The DSRCT involvement was found in the peritoneum, omentum, pelvis, organs in the abdominal cavity © 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd and retroperitoneum, lungs, lymph nodes, and bone marrow in both autopsied cases. They showed outstanding liver metastasis. The histopathological and immunohistochemical findings of these cases were also typical ones of DSRCT. It took more than 3 months to confirm the final diagnosis for Case 2. Initially, multiple nodules in the liver, lungs, bone, lymph nodes and peritoneum were regarded as metastatic and/or disseminated lesions from the pancreatic tumor, because a tumor of the pancreas tail was revealed by the first CT scan. The histologic findings of the hepatic needle biopsy, which contained only a small amount of tumor tissue, were largely consistent with hepatic metastasis of pancreatic carcinoma. The results of immunostaining were, however, atypical as pancreatic carcinoma and rather suggestive of DSRCT, though this suggestion was not strong enough to revise the clinical diagnosis. But, once we had gotten the split signal by FISH analysis, we soon reached the final diagnosis. It can be very important to consider an excisional biopsy if the histologic and/or immunohistologic findings of the hepatic needle biopsy specimen are not characteristic of metastatic carcinoma from abdominal organs. And if it is difficult to perform an excisional biopsy, it may be worth trying FISH analysis with the biopsied specimen for differential diagnosis of small round cell tumors.
The survival time after the initial presentation in Case 2 was much longer than that in Case 1, though both patients  had systemic metastases at the initial medical examination. Noteworthy is the 3.5-year survival after the definitive diagnosis in Case 2, because the survival time of metastasized DSRCT is approximately 2 years on average. Angarita et al. 5 reported that patients who had extra-abdominal metastasis had a significantly lower median OS than those without metastasis (20.7 months versus 28.8 months, P = 0.04).
The difference of administered therapies could be one of the reasons why the long-term survival was achieved in Case 2. In the former case, VAC-IE, GEM+DTX, and radiation therapies were used, but they could not achieve sufficient therapeutic effects. On the other hand, the tumor reduction effect was obviously observed on CT scans after FOLFIRINOX therapy in the latter case (Fig. 3a). Trabectedin, pazopanib and eribulin were administered after FOLFIRINOX therapy, but did not show obvious effects. FOLFIRINOX therapy is usually used in inoperable pancreatic cancer patients. 7 To the best of our knowledge, this was the first case in which FOLFIRINOX therapy was used for DSRCT. Further studies are needed in order to explain the therapeutic effects in Case 2.