Abstract
Primary primitive neuroectodermal tumor (PNET) of the cervix is extremely rare. Only few previous researches have reported on morphologic descriptions in frozen sections or changes in this tumor after radiotherapy or chemotherapy treatments. In the following study we investigated two cases of PNET in cervix. One of which had taken frozen exam and the other experienced neoadjuvant chemotherapy. Briefly, the morphological analysis in intraoperative frozen indicated small round cell malignant tumor with Homer-Wright rosettes, which similar to that in paraffin sections. The morphological changes after chemotherapy showed glial and epithelial differentiation as well as other common reaction. Immunochemistry test of neural and neuroendocrine markers and the detection of EWSR1 gene rearrangement may show positive in this disease in cervix. In conclusion, the obtained data may be useful for further examination, detection and diagnosis of PNET in clinical practice. Also, to the best of our knowledge, this was the first time that morphological and pathological changes were examined in PNET post-chemotherapy. However, the exact mechanisms of these changes require further exploration.
Keywords: Primitive neuroectodermal tumor, cervix, morphology, intraoperative frozen, neoadjuvant chemotherapy
Introduction
Primitive neuroectodermal tumor (PNET) originates from primitive neuroectodermal, and is a highly invasive tumor. PNET was initially described by Stout in 1918 [1], but it was not until 1930s that the concept of PNETs gained general recognition [2]. According to existing literature, cervical PNET occurred very rarely, and only sporadic cases have been reported [3-15]. In this study we reviewed 15 patients who had a relative detailed clinic-pathologic description, and we further analyzed two cases of primary cervical PNET in patients treated in National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, focusing on their morphologic and functional characteristics. We have obtain the informant consent from the patients.
Patients and methods
Case 1: a 39-year-old woman (gravida 3, para 1, abortus 2). The patient had no history of irregular vaginal bleeding and watery discharge; was diagnosed with multiple uterine leiomyoma, with the largest one observed in the cervix area. Tumor size (largest leiomyoma: 10.2×10.0×9.2 cm) and cystic degeneration were detected by computed tomography (CT) scan. Consequently, the large cystic leiomyoma in cervix were dissected and further analyzed by frozen sections, indicating a small round cell malignant tumor type. The patient received radical hysterectomy, bilateral adnexectomy and pelvic lymph node dissection. Additionally, the patient was treated with six cycles of chemotherapy (ifosfamide 2000 mg, cis-platinum 30 mg and doxorubicin 30 mg d1-3 ivgtt/q21 d). Since then, the patient has been on regular follow-up for 5 years without any signs of disease recurrence i.e. no evidence of disease (NED).
Case 2: a 14-year-old girl with vaginal irregular bleeding (1 month). A magnetic resonance imaging (MRI) scan of pelvis showed a space-occupying lesion in cervix (11.2×8.3×5.8 cm in size) and multiple swollen lymph nodes around the bilateral iliac blood vessels. Further cervical biopsy revealed PNET. After four cycles of neoadjuvant chemotherapy (NACT) (ifosfamide 2000 mg d1-2 iv, 1000 mg d3 iv and epirubicin 30 mg d1-3 iv/q21 d), the tumor could not be detected by ultrasound and MRI. Consequently, the patient refused subsequent chemotherapy and was placed on regular examination (every month). Two months later, a MRI scan of pelvis showed a recurrent mass in the cervix with a maximum diameter of 4.1 cm. Consequently, the patient received exploratory laparotomy, radical hysterectomy with pelvic and para-aortic lymphadenectomy, right salpingo-oophorectomy, and cystectomy. Since then, the patient has been on regular follow-up for 6 months with NED.
Results
In the case 1, the intraoperative frozen section showed that the tumor was composed of diffusely distributed small round malignant tumor cells with minimal cytoplasm and pepper-like nucleus chromatin (Figure 1A). In addition, Homer-Wright rosettes were frequently found within the tumor (Figure 1B). To conclude, the intraoperative frozen section diagnosis revealed “small round cell malignant tumor”.
Figure 1.
The tumors’ histological appearances. A, B. The intraoperative frozen section from patient one (Case I) showed that the tumor tissue was composed of diffusely distributed small blue-staining tumor cells with minimal cytoplasm and pepper-like nucleus chromatin, and Homer-Wright rosettes (H&E, ×400). C. The paraffin section of case one after operation (H&E, ×400). D, E. The tumor section of patient two (Case II) (previous to chemotherapy) was composed of small round cells with scanty cytoplasm with round nuclei containing fine chromatin (H&E, ×400). Homer-Wright rosettes were occasionally present (H&E, ×200). F-I. The tumor section of case two (post-chemotherapy) showed uniform small round cells with round nuclei (H&E, ×400). Obvious pathologic response, higher degree of neural differentiation, and cells with epithelial differentiation were observed post-chemotherapy (H&E, ×200).
The microscopic appearance of paraffin section was similar to the frozen slices, which also showed small round cell malignant tumor with Homer-Wright rosettes (Figure 1C). Immunohistochemistry (IHC) showed that tumor cells expressed CD99, FLI1, S-100, neuron specific enolase (NSE), CD56. However, no expression of pan-cytokeratin (AE1/AE3), synaptophysin (Syn), chromogranin A (CgA) were found. In addition, the Ki-67 proliferating index was about 30%. The fluorescence in situ hybridization (FISH) revealed EWSR1-FLI1 gene rearrangement in this case, confirming the diagnosis of PNET (Figure 2E).
Figure 2.
Immunohistochemical staining and FISH images. A. CD99 shows strong positive expression of cell membrane (200×). B, C. Fli-1 and CD56 show positive expression (200×). D. The Ki-67 proliferating index was 80% (200×). E. FISH of case one showing split signals (positive for EWSR1 rearrangement).
In the case 2, a cervical biopsy specimen showed the tumor was composed of small round cells with scanty cytoplasm and pepper-like nucleus chromatin (Figure 1D). Homer-Wright rosettes and pseudo-rosettes were occasionally present (Figure 1E). IHC showed positive expression of CD99, FLI1, and Vimentin, while AE1/AE3, GFAP, S-100 were negative. The tumor was diagnosed as PNET.
The post-operative specimen after NACT, showed some regions with uniform small round cells with pepper-like nucleus chromatin (Figure 1F). Furthermore, most regions of the tumor indicated obvious pathologic response to NACT, including foam cells aggregation, multinucleated giant cells proliferation, cholesterol crystals, and proliferating abnormal thick-walled blood vessels (Figure 1G). Additionally, some regions showed a high degree of glial like differentiation (Figure 1H). Other showed some epithelial differentiation in the border between the region of small round cell tumor and that of glial differentiation (Figure 1I). IHC revealed positive expression of CD99 (Figure 2A), FLI1 (Figure 2B), CD56 (Figure 2C), Syn. In addition, NF was focally positive, while all the other biomarkers were negative, including AE1/AE3, EMA, CK18, CK5/6, P63, P40, WT-1, ChrA, Desmin, SMA, MyoD1, Myogenin, and LCA. Moreover, Ki-67 proliferating index was approximately 80% (Figure 2D). FISH for EWSR1 gene rearrangement resulted negative for repeated detection.
Discussion
Two types of PNET i.e. central nervous system PNET and peripheral PNET were detected based on tumor location. Peripheral PNET may occur in lungs, kidneys, larynx and uterus, as well as the deep soft tissues of the lower extremities and paravertebral regions [16,17], while the PNET of the cervix is generally extremely rare. PNET of cervix was recognized for the first time in 1996 by Horn et al. [18] and Sato et al. [3], who identified its rosette like structures within the tumor tissue. To our knowledge, until the present day the presence of PNET in the cervix was only reported in 21 cases. In this study we reviewed 15 patients with relatively detailed clinic-pathologic descriptions, and we further analyzed 2 additional cases (Table 1).
Table 1.
Clinical and pathologic features, outcome of peripheral neuroectodermal tumors of the cervix
| Case no. | Author/year | Age (years) | Clinical features | Histologic features | Size | Follow-up | |||
|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||
| Irregular vaginal bleeding | Others | Small blue-staining tumor cells; scanty cytoplasm | Rosettes | Others | |||||
| 1 | Sato S et al. 1996 | 44 | + | + | + | 7 | Alive 6 months, NED | ||
| 2 | Tsao AS et al. 2001 | 24 | + | Urinary frequency | + | 8.9 | Alive 24 months, NED | ||
| 3 | Malpica A et al. 2002 | 35 | + | + | 4 | Alive 5 months, NED | |||
| 4 | Malpica A et al. 2002 | 50 | + | + | - | 3 | Alive 18 months, NED | ||
| 5 | Snijders-Keilholz A et al. 2005 | 21 | + | + | - | 5.5 | Alive 27 months, NED | ||
| 6 | Yang J X et al. 2006 | 59 | ①; bloody ascites | + | 3 | Died, 15 days | |||
| 7 | Farzaneh F et al. 2011 | 45 | ② | + | _ | 6 | Alive 4 years, NED | ||
| 8 | Arora N et al. 2012 | 23 | + | Dysuria; fibroid | + | ③ | 9.8 | Alive 4 years, NED | |
| 9 | Masoura S et al. 2012 | 23 | + | ① | + | - | ③; ④ (72/10 HPF) | 10 | Died, 12 days |
| 10 | Li B et al. 2013 | 27 | + | ①; ② | + | + | ④ | 5.5 | Alive at 6 months, NED |
| 11 | Khosla D et al. 2014 | 28 | + | ① | + | ④ (15/10 HPF) | 6 | Alive 33 months, NED | |
| 12 | Xiao C et al. 2014 | 52 | + | + | - | - | Pelvic recurrence 6 months, DOD 9 months | ||
| 13 | Xiao C et al. 2014 | 59 | + | + | - | - | DOD | ||
| 14 | Mashriqi N et al. 2015 | 49 | + | ① | + | ③; ④ numerous | 6.6 | Died, 10 months | |
| 15 | Weissferdt A et al. 2015 | 35 | + | ① | + | ③; ④ | - | Alive 1 years, NED | |
| 16 | Present case 1 | 39 | - | Fibroid | + | + | ④ | 10.2 | Alive five years, NED |
| 17 | Present case 2 | 14 | + | ② | + | + | ④; ⑤; ⑥ | 11.2 | Alive 6 months, NED |
Abbreviations: ①: Lower abdominal pain; ②: Vaginal discharge; ③: Necrosis; ④: Mitotic figures; ⑤: Pathologic response of chemotherapy; ⑥: Neuroectodermal differentiation; NED: no evidence of disease; DOD: died of disease.
PNET mainly occurred in adolescents or young adults (average age of 38). The most common symptoms were irregular vaginal bleeding and watery discharge, while the sizes of the lesions in the cervix varied from 3 cm to 10 cm, with the mean diameter of 6.3 cm (Table 1). Moreover, no treatment and follow-up guideline were reported. We further analyzed two cases: a 14 year old girl, which was also the youngest patient in this series, and 39-year-old woman who had space occupying lesion without other complains. Briefly, the neoplasms found in both cases were bigger than 10 cm, and their clinical properties were in accordance with the examined literature.
Microscopically, no difference in PNET morphology was observed in the tumor frozen slices as compared to paraffin slices. Briefly, the tissue was composed of diffusely distributed small round cells, with scanty cytoplasm and pepper-like nucleus chromatin, and was showing a lot of mitotic figures (Table 1). Moreover, Homer-Wright rosettes were observed within some well-differentiated tumors. Schmidt et al. affirmed that the presence of Homer-Wright rosettes was a diagnostic manifestation of PNETs [19], however these morphologic characteristics were observed in less than 50% of cases [11]. In this study, Homer-Wright rosettes were detected in both cases, which was in accordance with diagnostic description.
In the case 2, changes in pathologic response of cervical PNET were observed post-NACT. Common morphological changes, including foam cells aggregation, multinucleated giant cells proliferation, cholesterol crystals, and proliferating abnormal thick-walled blood, as well glial and epithelial differentiation were observed in cervical PNET post-NACT (Figure 1G-I). The glial differentiation showed pink-stained and a cord structure (Figure 1H). While the epithelial differentiation was detected at the border between the region of small round cell tumor and the region of glial differentiation (Figure 1I). These cells were arranged in nests, with a small nucleolus and broad cytoplasm. Moreover, to our knowledge, this was the first study that reported changes in cervical PNET post-NACT, which in turn shared some similarities with the immature teratoma in post-surgical tumor recurrence (immature tissues converted into mature tissues) [20]. However, the exact causes and significances were largely unknown, and require further research.
IHC analysis was essential for the diagnosis of PNET. High expression of CD99 on cell membrane was observed in almost all the cases. In addition, neural markers, such as NSE, NF were frequently and highly expressed. At least one neuroendocrine maker (such as CD56, Syn, CgA) was positive in this tumor. The observed characteristics of IHC expression in our two cases were in accordance with the literature. Furthermore, we suggested a panel of IHC stains of cervical PNET, including CD99, Fli-1, Vimentin, NSE, CD56, Syn, CgA, CK, LCA, Desmin, MyoD1 and Ki-67.
The EWSR1-FLI1 fusion gene was a characteristic chromosomal change of peripheral PNET [21]. EWSR1-FLI1 fusion gene was the result of t(11;22)(q24;q12) chromosome translocation. However, the detection of fusion gene had not been well accepted in clinical practice for cervical PNET (Table 2). We performed EWSR1-FLI1 fusion gene detection in both our two cases. The negative result in case 2 may be related to several factors, such as NACT. We suggested that the EWSR1-FLI1 fusion gene should be detected in the diagnosis of cervical PNET as much as possible.
Table 2.
Summary of immunohistochemistry results of peripheral neuroectodermal tumors of the cervix
| Case no. | Author/year | CD99 | NSE | CD56 | Syn | CgA | AE1/AE3 | Vim | NF | S100 | ki-67 | Molecular |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Sato S et al. 1996 | ND | + | ND | ND | - | ND | ND | ND | - | ND | ND |
| 2 | Tsao AS et al. 2001 | + | ND | ND | - | - | - | + | ND | ND | ND | ND |
| 3 | Malpica A et al. 2002 | + | ND | ND | Focally | - | - | ND | ND | ND | ND | ND |
| 4 | Malpica A et al. 2002 | + | ND | ND | Focally | - | ND | ND | ND | ND | ND | ND |
| 5 | Snijders-Keilholz A et al. 2005 | + | - | - | - | - | - | - | ND | ND | ND | ND |
| 6 | Yang J X et al. 2006 | + | + | ND | + | ND | - | ND | + | ND | ND | ND |
| 7 | Farzaneh F et al. 2011] | + | Focally | ND | + | + | - | ND | ND | ND | ND | ND |
| 8 | Arora N et al. 2012 | + | ND | ND | ND | ND | ND | - | ND | ND | ND | ND |
| 9 | Masoura S et al. 2012 | + | - | ND | - | - | - | + | ND | - | ND | EWSR1-FLI1 |
| 10 | Li B et al. 2013 | + | + | + | ND | ND | - | + | ND | - | 80% | ND |
| 11 | Khosla D et al. 2014 | + | + | ND | ND | ND | - | + | ND | - | ND | ND |
| 12 | Xiao C et al. 2014 | + | ND | ND | ND | ND | - | + | ND | - | ND | ND |
| 13 | Xiao C et al. 2014 | + | + | - | + | ND | - | ND | + | - | ND | ND |
| 14 | Mashriqi N et al. 2015 | + | + | + | + | ND | - | ND | ND | - | ND | EWSR1-FLI1 |
| 15 | Weissferdt A et al. 2015 | + | ND | Focally | + | - | - | ND | ND | - | ND | ND |
| 16 | Present case 1 | + | + | + | - | - | - | ND | ND | + | 30% | EWSR1-FLI1 |
| 17 | Present case 2 | + | ND | + | + | - | - | ND | Focally | ND | 80% | - |
Abbreviations: CD99: cluster of differentiation 99; NSE: neuron specific enolase; Syn: synaptophysin; CgA: chromogranin A; Vim: vimentin; NF: neurofilament; ND: not done.
Conclusion
PNET in cervix is a relatively rare occurring condition, more common in young patients. Clinical features of the cervical PNET are relatively nonspecific. If examination of cervix reveals uniform small round cell malignant tumor with Homer-Wright rosettes, PNET is one of the possible diagnosis. This is the first study that reported the morphologic changes of PNET in cervix after NACT. They were similar with the changes observed in the immature teratoma after recurrence. The glial and epithelial differentiation in cervix PNET after NACT may be very significant; nevertheless this issue requires further exploration.
Disclosure of conflict of interest
None.
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