Adult primary pulmonary primitive neuroectodermal tumor

Abstract

Primitive neuroectodermal tumors (PNET) arising directly from the lung are very rare but particularly aggressive neoplasms. We report a case of a 31-y-old man with primary pulmonary neuroectodermal tumor. We review the clinical as well as pathological features. As typical for these tumors, the diagnosis was initially delayed in our patient and prognosis was poor despite aggressive surgical resection, postoperative chemotherapy and local irradiation. Recent biological insights have revealed unique chromosomal translocations crucial to the pathogenesis of these tumors, most notably the EWS-FLI-1 translocation. We provide an overview of the molecular features of the Ewing Sarcoma Family of Tumors (ESFT) including PNET and their potential implications for therapeutic targeting.

Keywords: :

• Ewing sarcoma
• adult
• lung
• primitive neuroectodermal tumor

Introduction

Primitive neuroectodermal tumors (PNET) and Ewing sarcoma (EWS) belong to the same family of malignant, small, round-cell neoplasms of soft tissue or bone origin. Common locations for EWS-PNET include chest wall, pelvis and extremities. EWS-PNETs that arise in the lung parenchyma without chest wall involvement are extremely rare in adults. We report a case of EWS-PNET of the lung in an adult and review the distinct clinical, pathological and molecular features of these tumors.

Clinical Course

A 31-y-old Hispanic man presented with hemoptysis. His past medical history was remarkable for childhood asthma, a spontaneous pneumothorax and a remote history of smoking. At the time of his initial presentation in 2006, he was evaluated with chest radiography and a CT (CT) scan that showed a 2 cm nodule in the left lung, involving lingula. He underwent a fine needle biopsy of this nodule. However, histopathological examination was inconclusive showing only fibrosis. Fiberoptic bronchsoscopic biopsy was recommended to fully characterize this nodule. Unfortunately, the patient was lost to follow up until end of 2007 when presented with recurrent hemoptysis associated with left sided chest pain and fever. This time chest X-ray showed left lung opacity (Fig. 1), and a repeat CT scan of the chest revealed a well-defined dense mass in the lingula of the left lung measuring 10.7 × 7 × 11.4 cm (Fig. 2).

Figure 1. Chest radiogram of a 31-y-old man with primary pulmonary primitive tumor (PNET) in the left lung (lingula).

Figure 2. Computer tomographic scan of a 31-y-man with primary pulmonary primitive tumor (PNET) in the left lung.

A CT-guided core needle biopsy of this mass was inconclusive, but suggestive of a neoplasm. Histopathological examination showed fibrosis, with focal small-cell keratin-negative infiltrate. Immunohistochemical staining for keratin, EMA, S100, CD34, desmin were negative, positive for NSE and synaptophysin with an equivocal CD99 staining, suspicious for malignant small blue-cell neoplasm. To establish the final diagnosis, the patient underwent posterolateral thoracotomy with resection of the mass without lobectomy. Gross pathological examination showed a large mass in the linguala, which was adherent to the chest wall anteriorly and to the pericardium medially with extensive central necrosis and hemorrhage (Fig. 3). The pleural surface was smooth, pink and focally congested with a few adhesions. The soft tissue overlying the mass was congested with diffuse, red adhesions. For diagnosis confirmation, the tissue was evaluated for t(11:22) translocation. Final results showed positive staining for CD99 (Fig. 4) and a chimeric fusion translocation for EWSR1-ERG consistent with pulmonary EWS-PNET. Post-surgery follow-up CT chest showed a residual opacity that was 5.8 cm in transverse, and 4.7 cm in antero-posterior, 9.5 cm in craniocaudal dimensions. These radiographic changes were interpreted as postoperative in nature, although the persistent tumor could not have been completely excluded. There was no mediastinal lymphadenopathy. There were no lytic or blastic osseous lesions noted on chest CT scan, with the exception of posterior left seven fractures from thoracotomy and a whole-body nuclear bone scan showed no scintigraphic evidence of bone metastasis. A CT scan of the abdomen and pelvis showed no evidence of metastatic disease. Following the completion of staging studies, the decision was made to start aggressive systematic chemotherapy and adjuvant RT considering the inability to obtain clear surgical margins, as well as the high probability of recurrence. The chemotherapy regimen consisted of alternating cycles every 3 wk of cyclophosphamide, doxorobucin and vincristine, with isofosfamide and etoposide, respectively, in conjunction with growth factor support.

Figure 3. Well demarcated primary pulmonary primitive tumor (PNET) with extensive tumor necrosis inside.

Figure 4. HH&E stained section of a small round cell lung tumor-primary pulmonary tumor (PNET).

After completing four cycles of chemotherapy, the patient started external beam irradiation therapy of the left hemi-thorax including: chest wall, pericardium and hilum, which were completed within 6 mo after surgery. He completed a total of 11 cycles of chemotherapy over the period of 1 y. He underwent serial CT chest for restaging that showed stable left lingular mass with no evidence of recurrent lung malignancy. Hence, during this time, he continued to have progressive worsening of chest pain at the thoracotomy site, and recurrent lung infections. Two months later following chemotherapy, CT of the chest showed a left-side pleural mass that was increasing in size, a left pleural effusion and a new right middle lung nodule. A core needle biopsy of the pleural mass confirmed that tumor relapsed.

He had progression of his disease with development of lesions at both the chest wall and within the lung parenchyma despite trying multiple palliative chemotherapy regimens that included cyclophosphamide, topotecan, carboplatin and gemcitabine, radiotherapy and docetaxel and patient succumbed to his disease after 3 y.

Discussion

In 1918, Stout et al.¹ reported an extracranial PNET originating in the ulnar nerve of an adult patient. Ewing sarcoma (EWS), initially named diffuse endothelioma, was first described in 1921 by James Ewing, who reported a case of highly malignant tumor in the diaphysis of long bones in a 14-y-old girl.² In 1979, Askin et al.³ described a similar rare malignant small-cell tumor of the thoracopulmonary region in 20 children and adolescents with a mean age of 14 y, referred to as Askin’s tumor. ES, extra-osseous Ewing tumors (EES), PNET and Askin’s tumors (PNET of the chest wall) have been categorized as members of the Ewing’s sarcoma family of tumors (ESFT). Most commonly seen in children and young adults, these highly malignant tumors of bone and soft tissue are histopathologically described as poorly differentiated “small round cell tumors.” ESFT are tumors of neural crest derivation showing varying degrees of neuroectodermal differentiation.⁴ Current evidence indicates that ES and PNET represent a single neoplastic entity, differing only in their degree of neural differentiation. Tumors that demonstrate neural differentiation by these analyses have been traditionally named PNETs, and those that are undifferentiated have been called Ewing's sarcoma.⁵

Clinical symptoms depend on the site of presentation but invariably include pain and swelling of the surrounding structures due to mass effect. Other reported symptoms and signs are site specific, such as hemoptysis with lung involvement. Definitive diagnosis of primary pulmonary EWS-PNET is based on a broad combination of imaging tests and information obtained with light microscopy, immunohistochemical staining and cytogenetic analysis. Imaging tests include chest radiographs, CT scans and MRI to determine the extent of tumor involvement. On CT scans, EWS-PNETs appear as heterogeneous masses, often invading surrounding tissues, including bone. MRI typically reveals a mass isointense to muscle on T1-weighted images, while hyperintense on T2-weighted images. Most of the tumors located in the chest show necrosis and multilocular cysts, while some of the tumors appear as homogeneous solid masses.⁶ Imaging tests can be used to guide biopsy of identified masses. On light microscopy, EWS-PNETs appear as small round blue cells with hyperchromatic nuclei and scant cytoplasm, associated with extensive necrosis. Immunohistochemically, EWS-PNETs typically co-express CD99 (the glycoprotein MIC2) and vimentin. MIC2 expression has a high sensitivity for diagnosis of EWS-PNET family of tumors, but it lacks specificity in that many other tumors express MIC2.⁷ Other nonspecific markers include S-100, neuron-specific enolase, CD75 and synaptophysin. Because PNETs are uncommon yet highly aggressive, a high index of suspicion is required for timely diagnosis and prompt initiation of treatment. The treatment of PNETs should be aggressive consisting of early surgical resection followed by a combination of intense chemotherapy and high-dose radiation therapy. The most compelling evidence suggests chemotherapeutic five-drug regimens that include alternating cycles of vincristine, doxorubicin and cyclophosphamide with ifosfamide and etoposide. It has been shown that patients with loco-regional disease have an improved survival with dose-intensified chemotherapy.⁸ Prognosis is mainly related to the ability to achieve disease-free surgical margins and the extent of anatomical spread to surrounding structures such as bone, pleura, and the epidural space. Our patient had pleural involvement without bone extension. Historically, outcome has been poor because of the limitations of traditional chemotherapeutic approaches.⁹ This case highlights the importance of early diagnosis and treatment of primary pulmonary PNETs. It also demonstrates the shortcomings of traditional chemotherapeutic agents in altering the aggressive natural history of these tumors. In the last few years, emerging insights into the molecular biology of these tumors have opened the door to the development of targeted therapies needed to improve the prognosis of these rare aggressive tumors.

Molecular biology of ESFTs

ESFTs are characterized by a specific balanced chromosomal translocation which leads to classification of these tumors as a single entity with a spectrum of phenotypic differentiation. The most common and studied fusion genes are EWS and FLI1. These translocations interrupt specific genes and recombine them to create novel fusion genes that encode tumor-specific proteins (Fig. 5).¹⁰

Figure 5. Schematic diagram of EWS-FLI1 gene fusion resulting from t(22;11) translocation. The TET family associated RRM domain, the ETS DNA-binding domain (ETS-DBD) and the N-terminal transactivation domain (ATA) are shown. The fusion gene can vary depending on whether exons 5–9 or 6–9 of FLI1 are included. Adapted with permission from ref. 10.

Ewing sarcoma (EWS) gene and protein

The EWS gene is located on band q12 of human chromosome 22, it spans about 40 kb of DNA encoding 17 exon.^11,12 The EWS protein is part of the TET family of proteins, together with the Translocated in liposarcoma (TLS) and TATA-binding protein-associated factor 15.¹³ TET proteins share a characteristic 87-amino acid ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM) domain that plays a key role in protein-RNA binding.¹⁴ On one end, TET proteins possess a glutamine rich N-terminal region that fuses to ETS genes in ESFT. On the other end, at the C terminal, TET proteins have a distinctive region with a variable number of RGG (arginine-glycine-glycine) residues thought to mediate interactions with RNA or single-stranded DNA.¹⁵ Functionally TET proteins are involved in transcription and mRNA processing by binding components of both transcription and splicing regulators. EWS is ubiquitously expressed in mammalian cells and binds to transcription products of RNA polymerase II, thus having a pivotal role in basic transcriptional regulation.¹⁶

Friend leukemia integration 1 (FLI-1) gene and protein

FLI-1 gene is a member of the ets family of genes. FLI-1 maps to the mouse chromosome region syntonic with human chromosome 11q23.¹⁷ This is mainly expressed in hematopoietic cells, both during development and in adult tissues with a lower expression levels in non-hematopoietic adult tissues, such as lung, heart and ovaries. It was identified proximal to the insertion of Friend’s murine leukemia virus as well as other viruses.^18,19 The human FLI-1 gene codes for a 452-residue protein consisting of two domains separated by a FLI-1-specific domain (FLS); the 3′ region encodes for an 89-amino acid carboxyterminal trans-activation (CTA) domain involved in DNA binding; the 5′ ets and FLS regions encode for N-terminal transcriptional activation (ATA) domain.^20,21 FLI-1 plays a pivotal role in the regulation of hematopoiesis and vasculogenesis. FLI-1 affects the normal balance between differentiations and proliferation in primary erythroblasts, inducing malignant transformation; the main mechanism is induction of Bcl2 expression and further enhancement of cell survival. In addition, FLI-1 inhibits the expression of retinoblastoma protein at the transcriptional level, further promoting the G₁ and S transitions of the cell cycle.²²

Unique genetic translocations

A common genetic abnormalities involving fusion of the EWS gene on chromosome 22 and one of the several members of the ETS family genes constitute the genetic hallmark of ESFTs. In 85% of cases, the gene fusion is the result of a balanced chromosomal translocation between EWS gene and FLI-1 t(11;22)(q24;q12).²³ In 5–10% of cases 10% of EWS-PNETs, there is a gene fusion resulting from translocation between EWS and ERG (21q22), which is another member of the ETS family of transcription factors closely related to FLI1.²⁴ Several other translocations are rarely seen. EWS-ERG was initially thought to be a simple balanced reciprocal translocation; however, further investigation using systematic analysis with locus-specific probes identified the breakpoints within chromosome 21 and 22. These breakpoints are required for the excision and exchange of both 3′ERG and 3′EWS, that results in the formation of the EWS-ERG fusion gene present on the der(22).²⁵ Additional acquired chromosomal and genomic abnormalities were observed also, highlighting the complex chromosomal rearrangements of EWS-ERG Ewing sarcoma (ES) gene. Combinations of EWS and ETS family drive ontogenesis in ESFTs. EWS rearrangements with other genes result in different types of sarcomas emphasizing that it is the partner gene, not EWS that drives the final tumor pathology. In fact, rare fusions that do not even involve EWS (CIC/DUX4, BRD4/NUT) were found to induce similar molecular changes to EWS/ETS fusions. A new rare translocation that fuses EWS to NFATc2 was also identified.²⁶

EWS-FLI-1 fusion protein

The EWS-FLI1 fusion transcript encodes an oncoprotein with two primary domains, the N-terminal domain of EWS, which is a potent transcriptional activator and the C-terminal region of FLI1, which harbors the DNA binding domain.²⁷ EWS-FLI1 and other EWS-ETS fusions are multifunctional: activating or repressing multiple target genes.²⁸ The list of both direct and indirect genomic and proteomic targets for EWS-FLI1 is growing; one of the most critical targets is transcription factor NKX2–2, which acts as a transcriptional repressor. EWS-FLI-1 upregulates NKX2–2 mediating a downregulated transcriptional signature that results in oncologic transformation. Both EWS-FLI1 domains are important for oncogenic transformation of target cells, as deletion of either domain results in complete loss of oncogenic activity.^29,30

Diagnostic and prognostic implications

Many molecular techniques can be used to detect translocations associated with EWS-PNETs, including fluorescent in situ hybridization, Southern blotting, DNA-based polymerase chain reaction (PCR), RNA-based PCR, also called reverse transcriptase-PCR (RT-PCR). The mainstay in molecular diagnosis is RT-PCR, because of high sensitivity and specificity.³¹ Better outcomes were observed in patients with localized tumors that express the most common translocation, EWS exon 7 fused to FLI1 exon 6, compared with other less common fusion types.³² The heterogeneity in EWS-PNETs prognosis correlates with molecular heterogeneity that resulting from changes either in the fusion partner (FLI1, ERG, ETV1, E1A, or FEV), or the breakpoint location within the genes. TP53 mutation, del1p, homogenous deletion of CDKN2A is associated with poor prognosis.³³

EWS-FLI-1 as a therapeutic target

The limitations of traditional chemotherapeutic approaches have fueled the search for novel targeted therapies to improve clinical outcomes. The elucidation of the EWS-FLI1 translocation and its cellular functions presents a unique opportunity for therapeutic targeting. EWS and ETS fusions play a pivotal role in ESFTs’ pathogenesis, thus referred to as the “perfect target without a therapeutic agent.”³⁴ Different therapeutic strategies targeting EWS-FLI1 are currently under investigation. In vitro experiments show that simultaneous administration of EWS/FLI-1-targeted antisense oligonucleotides and rapamycin induces apoptosis of ES cells. In vivo experiments with the antisense/rapamycin combination show inhibition of the growth of the ESFT xenografts in mice.³⁵

Another promising strategy involves the use of small interfering RNA (siRNA). An siRNA molecule was designed with an aromatic compound at the 3′-end to target the breakpoint of EWS/Fli1 fusion gene. In vitro experiments showed that in ES cells, this siRNA molecule significantly suppresses the expression of the EWS/Fli1 protein sequence and concomitantly reduces c-Myc protein expression. The inhibition of proliferation of ES cells was further demonstrated in vivo by using Ewing sarcoma cell line subcutaneously xenografted into mice.³⁶ More research is needed on the safety and efficacy of these novel targeted approaches for treatment of ESFT in humans.

Conclusions

We present the rare case of a patient with pulmonary PNET, a tumor from the ESFTs. Based on the small number of cases (Table 1), PNETs are rare aggressive neoplasms with a very high rate of local recurrence. Despite aggressive therapy, our patient rapidly succumbed to the tumor. Improving understanding of the molecular pathogenesis underlying ESFTs especially the EWS-FLI1 translocation presents unique opportunities for therapeutic targeting. More research is needed to translate these basic discoveries from the bench to therapies that would result in clinical benefit for patients afflicted with these aggressive malignant soft tissue tumors.

Table 1. A literature summary of case reports of Primary Primitive Neuroectodermal Tumor (PNET) of the lung/chest wall

Authors - journal - year	Title
1. Burge HJ, Novotny DB, Schiebler ML, Delany DJ, McCartney WH, Chest. Vol 97, 1252–1254, 1990	MRI of Askin’s tumor. A case report at 1.5 T.
2. Cabezali R, Lozano R, Bustamante E, Castiella T, Güemes A, Ramírez J, Moncada E et al., Journal of Thoracic and Cardiovascular Surgery. Vol 107, 960–962, 1994	Askin’s tumor of the chest wall: a case report in an adult.
3. Imamura F, Funakoshi T, Nakamura S, Mano M, Kodama K, Horai T, Lung cancer, Vol 27, 55–60, 2000	Primary primitive neuroectodermal tumor of the lung: report of two cases.
4. Baumgartner FJ, Omari BO, French SW, Ann Thorac Surg, Vol 72, 285–287, 2001	Primitive Neuroectodermal Tumor of the Pulmonary Hilum in an Adult.
5. Kahn AG, Avagnina A, Nazar J, Elsner B, Arch Patho Med, Vol 125, 397–399, 2001	Primitive neuroectodermal tumor of the lung.
6. Paik SH, Park JS, Koh ES, Kim HK, Shin HK, Hong HS et al., European Radiology, Vol 16,2138–2131, 2006	Primary pulmonary primitive neuroectodermal tumor: CT and skeletal scintigraphic image features with pathologic correlation.
7. Lee YY, Kim DH, Lee JH, Choi JS, In KH, Oh YW et al. J Korean Med Sci, Vol 22, 159–163, 2007	Primary Pulmonary Ewing's Sarcoma/Primitive Neuroectodermal Tumor in a 67-y-old Man.
8. Verma S, Prakash P, Yadav P, Srivastava D, Sharma SC, JIACM, Vol 9,237–239, 2008	Primitive Neuroectodermal Tumour of the Chest Wall in an Adult.
9. Antelo JS, García CR, Martínez CM, Hernando HV, Arch Bronconeumol, Vol 46, 44–46, 2010	Pulmonary Ewing Sarcoma/Primitive Neuroectodermal Tumor: A Case Report and a Review of the Literature.
10. Gedik GK, Sari O, Altinok T, Tavli T, Kaya B, Kara PO, Case Report in Medicine, Article ID 517329, 2009	Askin's Tumor in an Adult: Case Report and Findings on 18F-FDG PET/CT.
11. Gaude GS, Malur PR, Kangale R, Anurshetru S, Lung India, Vol 26, 89–91, 2009	Primitive neuro-ectodermal tumor of the lung in an adult.
12. Guade GS, Patil P, Mlur P, Annurshetru S, Journal Of Thoracic Disease, Vol 3, 2011	Premitive neuro-ectodermal tumor of the lung.
13. Weissferdt A, Moran CA, Lung, July 17, 2012	Primary Pulmonary Primitive Neuroectodermal Tumor (PNET): A Clinicopathological and Immunohistochemical Study of Six Cases.

Abbreviations:
PNET	=	primary primitive neuroectodermal tumor
ES	=	Ewing’s sarcoma
EES	=	extra-osseous Ewing tumor
ESFT	=	Ewing’s sarcoma family of tumors
CT	=	computed tomography
RT	=	radiotherapy
PCR	=	polymerase chain reaction
RT	=	reverse transcriptase
siRNA	=	small interferering RNA
TLS	=	translocated in liposarcoma
FLI-1	=	Friend leukemia integration 1

Acknowledgments

The authors wish to thank Blackman J. for his assistance with figures in the manuscript.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.