http://ijs.sagepub.com/ International Journal of Surgical Pathology http://ijs.sagepub.com/content/22/5/427 The online version of this article can be found at: DOI: 10.1177/1066896913502227 2014 22: 427 originally published online 17 October 2013 INT J SURG PATHOL Damasceno, Pedro Bastos and Manuel Sobrinho-Simões Helena Barroca, Conceição Souto Moura, José Manuel Lopes, Susana Lisboa, Manuel R. Teixeira, Margarida PNET with neuroendocrine differentiation of the lung: Report of an unusual entity Published by: http://www.sagepublications.com can be found at: International Journal of Surgical Pathology Additional services and information for http://ijs.sagepub.com/cgi/alerts Email Alerts: http://ijs.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: http://ijs.sagepub.com/content/22/5/427.refs.html Citations: What is This? - Oct 17, 2013 OnlineFirst Version of Record - Jul 20, 2014 Version of Record >> at b-on: 01100 Universidade do Porto on July 28, 2014 ijs.sagepub.com Downloaded from at b-on: 01100 Universidade do Porto on July 28, 2014 ijs.sagepub.com Downloaded from
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http://ijs.sagepub.com/International Journal of Surgical Pathology
http://ijs.sagepub.com/content/22/5/427The online version of this article can be found at:
DOI: 10.1177/1066896913502227
2014 22: 427 originally published online 17 October 2013INT J SURG PATHOLDamasceno, Pedro Bastos and Manuel Sobrinho-Simões
Helena Barroca, Conceição Souto Moura, José Manuel Lopes, Susana Lisboa, Manuel R. Teixeira, MargaridaPNET with neuroendocrine differentiation of the lung: Report of an unusual entity
Published by:
http://www.sagepublications.com
can be found at:International Journal of Surgical PathologyAdditional services and information for
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Ewing’s sarcomas/primitive neuroectodermal tumors (PNETs) affect young adults and are small round cell tumors, characteristically expressing CD99 and FLI1 and presenting a nonrandom translocation involving the EWSR1 gene (or rarely the FUS gene) and a member of the ETS family of genes.1 In 90% of the cases, the trans-location involves the EWSR1 and the FLI1 genes.2,3 Recently, numerous histological variants (adamantinoma-like, large cell, spindle cell, sclerosing, clear cell, and vascular-like) as well as cases displaying atypical mor-phological differentiation (eg, epithelial differentiation) or infrequent immunostaining features (eg, epithelial markers) have been described in Ewing’s sarcoma/PNET tumors.4,5 These variants are not linked to the type of translocation partner. 6-8 Epithelial expression is reported in 20% of the cases, generally focal, not exceeding 10% of tumor cells, and restricted mainly to low-molecular-weight cytokeratin in some Ewing’s sarcoma/PNET tumor variants, namely the adamantinoma-like subtype.6,7,9,10 Neuroendocrine markers, such as neuron-specific enolase
and synaptophysin, have been reported in Ewing’s sar-coma/PNET tumors, but to our knowledge, chromogranin immunoexpression in such tumors is exceedingly rare.11-13
We report a case of an unusual lung primary tumor in a 20-year-old female, morphologically similar to a classic Ewing’s sarcoma/PNET, tumor in which there are foci of squamous differentiation, intense, and diffuse (membrane) expression of CD99, high-molecular-weight keratins, p63
502227 IJSXXX10.1177/1066896913502227International Journal of Surgical PathologyBarroca et alresearch-article2013
1Centro Hospitalar S João, Porto, Portugal2Departamento de Patologia e Oncologia Médica da Faculdade de Medicina da Universidade do Porto, Porto, Portugal3Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal4Instituto Português de Oncologia do Porto e Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal
Corresponding Author:Helena Barroca, MD, Serviço de Anatomia Patológica, Centro Hospitalar de S João, Alameda Hernani Monteiro, 4200-465 Porto, Portugal. Email: [email protected]
PNET with neuroendocrine differentiation of the lung: Report of an unusual entity
Helena Barroca, MD1,2, Conceição Souto Moura, MD1,2, José Manuel Lopes, MD, PhD1,2,3, Susana Lisboa, MSc4, Manuel R. Teixeira, MD, PhD4, Margarida Damasceno, MD1,2, Pedro Bastos, MD, PhD1, and Manuel Sobrinho-Simões, MD, PhD1,2,3
AbstractEwing’s sarcoma/primitive neuroectodermal tumor (PNET) has been the subject of recent reports describing morphologic variants (adamantinoma-like, large cell, spindle cell, sclerosing, clear cell, and vascular-like) of the most classic form, as well as cases displaying unusual morphologic differentiation and atypical immunohistochemical features. We report a case of an uncommon lung tumor in a 20-year-old female, morphologically and molecularly consistent with an Ewing’s sarcoma/PNET tumor with foci of squamous differentiation, and peculiar expression of vimentin, high-molecular-weight keratins, p63, synaptophysin, and chromogranin. This case raises a challenging differential diagnostic problem with therapeutic implications: Should the patient be treated following the protocols for Ewing’s sarcoma/PNET tumors or as for lung carcinoma with neuroendocrine features? The patient we report here was treated with neoadjuvant chemotherapy for Ewing’s sarcoma/PNET according to Euro Ewing 99 study protocol followed by surgery and has no evidence of disease 15 months after the initial diagnosis. This highlights the importance of achieving the correct diagnosis of these atypical tumors using all clinical, morphological, and ancillary methods available to allow for their correct and timely treatment.
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and synaptophysin, and focal expression of chromogranin, as well as the presence of the EWSR1/FLI1 fusion gene.
Case Report
In September 2011, a 20-year-old female was admitted in a regional hospital because of recurrent episodes of pneu-monia in the left lung. Her clinical condition deteriorated and in March 2012, a bronchoalveolar lavage was per-formed. The cytological diagnosis of small cell neuroen-docrine carcinoma was suggested (Figure 1).
The patient was referred to our institution and in a positron emission tomography–computed tomography scan, a primary left pulmonary hilar mass (72.6 mm largest dimension) was identified (Figure 2). Multiple nodular opacities with sizes ranging from 6 to 12 mm were also identified in the left lung. A surgical bronchial biopsy disclosed a poorly differentiated small round cell tumor (Figure 3). Periodic acid–Schiff stain-ing without and with diastase treatment disclosed the pres-ence of intracytoplasmic glycogen in the neoplastic cells, by immunohistochemistry the tumour cells expressed CD99 (membrane positivity), synaptophysin, Cam 5.2, AE1AE3, keratin 5, chromogranin and p63, in the absence vimentin, CD56 (N-CAM), TTF1, Bcl2, and desmin expression. The tentative diagnoses of small cell neuroendocrine carcinoma, poorly differentiated synovial sarcoma, and desmoplastic small round cell tumor were ruled out and a diagnosis of Ewing’s sarcoma/PNET tumor was suggested. The patient started neoadjuvant chemotherapy for Ewing’s sarcoma/PNET according to Euro Ewing 99 study protocol with VIDE (vincristine, isofosfamide, doxorubicin, and etoposide).
Five months later, a positron emission tomography–computed tomography scan disclosed a remaining lung hilar lesion with 36.2 mm and the disappearance of pre-treatment-identified nodular opacities. A sleeve resec-tion of the left lower lobe (lower lobe 141gr) was performed; preoperative frozen-section examination of the divided edges showed clear and adequate margins. On macroscopy, a tumor (35 × 20 × 18 mm) involving the lobar bronchus was identified. The tumor was com-posed of irregular nests of small poorly differentiated round cells with nuclei displaying fine chromatin and inconspicuous nucleoli (Figure 4). Few dispersed foci of anaplastic cells (Figure 5A) were seen in addition to sharply limited foci of swirling large cells suggesting squamous differentiation (Figure 5B). Focally, rosette-like structures were identified. The tumor invaded the cartilage of the proximal bronchus, and disclosed numerous images of perineural and vascular invasion (Figure 5C and D).
Three months after surgery, the patient had no evidence of disease and started 3-dimensional conformal radiotherapy.
Immunohistochemistry
Immunohistochemistry evaluation was performed on the biopsy and on the specimen (Table 1). Immunostains were performed on formalin-fixed, paraffin-embedded sections after retrieval using standard avidin–biotin immunoperox-idase detection technique with appropriate positive and negative controls in a Roche Ventana system (Tucson, AZ). The monoclonal antibodies used and their manufac-turers are listed in Table 1.
Figure 1. Bronchial wash: Small cells with hyperchromatic nuclei. Two cylindrical ciliated epithelial cells can also be seen at the bottom (hematoxylin and eosin, 600×).
Figure 2. Initial positron emission tomography–computed tomography scan evaluation: Left pulmonary hilar mass measuring 72.6 mm.
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Almost all neoplastic cells were strongly positive for CD99 (membrane staining), synaptophysin, AE1/AE3, keratin 5, and p63 (Figure 6). Focal immunoexpression for chromogranin, EMA, and CEA were present in the absence of TTF1, keratin 7, Bcl2, desmin, CD56 (NCAM), S100 protein, GFAP, vimentin, calcitonin, and NUT expression. Table 1 summarizes the results of the immunohistochemi-cal study.
Fluorescence In Situ Hybridization Analysis
Dual-color fluorescence in situ hybridization (FISH) analy-sis was performed on paraffin-embedded tissue sections in the surgical specimen, using a commercial probe flanking the EWSR1 gene (Vysis LSI EWSR1 Dual-Color Break Apart Probe, Abbott Molecular, Des Plaines, IL) and BAC generated probes (BACPAC Resources Center, Oakland,
Figure 3. Surgical bronchial biopsy (A, hematoxylin and eosin, 100×; B, hematoxylin and eosin, 400×): Small cell tumor with crushing artifact.
Figure 4. The tumor was highly aggressive invading the cartilage of the main bronchus (A, hematoxylin and eosin, 40×), and highly cellular, comprising nests of small neoplastic cells with high N/C ratio, regular nuclei with fine chromatin and inconspicuous nucleoli (B, hematoxylin and eosin, 100×).
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Figure 5. (A) Anaplastic cells were found occasionally; (B) “Abrupt” foci of malignant cells with more abundant and dense cytoplasm organized in a swirling pattern, suggesting squamous differentiation. (C) Vascular invasion (D) Perineural invasion.
Table 1. List of Monoclonal Antibodies Used and Manufacturers: Immunohistochemical Results in the Specimen and in the Biopsy.
CA) flanking the FLI1 gene (RP11-1079A6 and RP11-69E19). The hybridization results were observed using a Zeiss Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) and images were captured with a Cohu 4900 CCD camera and a CytoVision system version 3.9 (Applied Imaging, Santa Clara, CA).
Fluorescence in situ hybridization analysis with the Vysis LSI EWSR1 Dual Color Break Apart Probe showed the pres-ence of a rearrangement involving the EWSR1 gene, and FISH analysis using BAC generated probes flanking the FLI1 gene exhibited separation of the 5′ and 3′ FLI1 probes, indi-cating the presence of a rearrangement involving the FLI1 gene. The combined FISH findings indicate the presence of the EWSR1-FLI1 fusion gene, characteristic of the Ewing’s sarcoma/PNET tumors (Figure 7). This rearrangement was homogeneous across morphologically different tumor areas.
Figure 6. Periodic acid–Schiff stain disclosing cytoplasmic glycogen, digested after diastase treatment, in tumor cells. Strong and diffuse CD99 (membrane), CK5, P63, and AE1AE3 expression displayed by the tumor cells; note also the diffuse synaptophysin (no architectural variation of expression was remarked), and the focal chromogranin, EMA, and CEA expression.
Figure 7. (A, B) EWSR1 A and FLI1 B: Fluorescence in situ hybridization (FISH) analysis. Dual color Break Apart probe (Abbott) on a paraffin-embedded tissue section indicates the presence of a rearrangement involving the EWSR1 gene in A and a rearrangement involving the FLI1 gene in B.
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Discussion
The recognition of the existence of unusual morphological patterns and unusual immunophenotypical expression in the Ewing’s sarcoma/PNET tumor hampers the differential diagnosis with other tumors in the age-group of the case reported here.1,14 Indeed, other small round cell tumors, namely alveolar rhabdomyosarcoma, poorly differentiated synovial sarcoma, desmoplastic small round tumor, myo-epithelial carcinoma, neuroendocrine carcinoma, and poorly differentiated carcinoma harboring the NUT fusion gene, should be considered in the differential diagnosis. In the setting of primary small cell bone neoplasms, epithelial and neuroendocrine pheatures have been reported previ-ously in tumours designated as polyhistiomas and primi-tive multipotential primary sarcomas.15,16 Most of the aforementioned tumors share the morphological character-istics of the Ewing sarcoma/PNET tumors and some dis-play CD99 positivity as the ESWR1 gene rearrangement.17-20 However, to our knowledge, no reliable study has so far reported the presence of the EWSR1/FLI1 gene fusion in tumors other than Ewing’s sarcoma/PNET tumors.
The case we have reported here represents an unusual tumor with features not previously described. In our case, the absence of vimentin, desmin, S100 protein, GFAP, Bcl2, and NUT expression were useful to rule out the less probable hypotheses of rhabdomyosarcoma, desmoplastic small cell tumour, myoepithelial carcinoma, synovial sar-coma, and poorly differentiated carcinoma (NUT positive) of the midline. Despite the presence of synaptophysin and chromogranin expression, the absence of TTF1, CD56 (NCAM), calcitonin, as well as the diffuse and intense expression of p63 reduced the possibility of a (primary or secondary) small cell neuroendocrine carcinoma of the lung. Moreover, the morphological features of the herein reported tumor did not fit within the classical framework of a poorly differentiated neuroendocrine lung carcinoma (ie, small cell lung carcinoma [SCLC] or neuroendocrine large cell carcinoma [NLCC]). In addition, poorly differ-entiated neuroendocrine lung carcinomas, both SCLC and NLCC, are rare in young patients, although a few cases have been reported.21 It is also not expected that a patient with SCLC or a NLCC of the lung would carry such a long follow-up, alive with no disease at the moment of the pres-ent report, 15 months after the first diagnosis.
The presence of small foci of squamous differentiation as well as the diffuse expression of p63 and high molecular weight keratins together with the chromogranin expres-sion raised the possibility of a poorly differentiated carci-noma with morphological and immunohistochemical features of squamous differentiation and focal neuroendo-crine differentiation. The EWSR1/FLI1 fusion gene was nevertheless homogeneously present across the histologi-cally different tumor areas and therefore most likely
preceded the morphological divergence observed. After an exhaustive literature search we did not find any report linking the presence of t(11;22)(q24;q12) to poorly differ-entiated carcinomas or to neuroendocrine carcinomas.
Weinreb et al8 report a unique case of a small round cell tumor, morphologically similar to our case. Both tumors show “abrupt” keratinization within a monotonous small round cell pattern, and both tumors express intense and diffuse expression of CD99, p63, and high-molecular-weight keratin CK5/6. Both tumors also present the EWSR1/FLI1 fusion gene. As in our case, and despite the absence of the characteristic morphologic pattern previ-ously described in the adamantinoma-like Ewing sarcoma/PNET tumor variant (associated to prominent desmoplas-tic tumor response, neoplastic nests with peripheral pali-sading, and basaloid morphology), Weinreb et al,8 based on the presence of the t(11;22) translocation and on the extensive morphological and immunohistochemical epi-thelial differentiation, classified the tumor as an adamanti-noma-like Ewing’s sarcoma/PNET tumor. Unlike previous reported cases of adamantinoma-like Ewing’s sarcoma/PNET tumor, neither our case nor that of Weinreb et al8 was located in the extremities; nor did it have any relation-ship with bone. Weinreb et al used, as additional reason for their classification, the claim that since the tumor was incompletely excised, other putative patterns, namely the adamantinoma-like, could have been left behind.
Besides the differences between adamantinoma-like Ewing’s sarcoma/PNET tumor and the case reported by Weinreb et al,8 as well as the present case, the expression of chromogranin A and synaptophysin in our case adds an extra level of diagnostic complexity. Taking all the data on record into consideration, we realize that the peculiar find-ing of neuroendocrine carcinoma features in a tumor with the classic EWSR1/FLI1 rearrangement is rare but it does occur. In fact, a recent report describes 2 thyroid Ewing’s sarcoma/PNET tumors confirmed by strong CD99 expres-sion and EWSR1 rearrangement, one of which being strongly positive for synaptophysin expression and weakly positive for chromogranin.13 In such cases, it is difficult to answer to the clinician’s question: How should these patients be treated? Should the therapy be similar to classic Ewing’s sarcoma/PNET tumors albeit displaying features that come close to other sarcoma or carcinoma histotype?
The case reported here represents an uncommon tumor sharing some of the major features of Ewing’s sarcoma/PNET tumor (morphologic pattern, glycogen rich neoplas-tic cells, CD99 expression and typical t(11;22)) and some peculiarities not previously reported in Ewing’s sarcoma/PNET tumor, namely the absence of vimentin, the strong and diffuse immunoexpression for high-molecular-weight keratins and p63, and the focal chromogranin expression. Primary Ewing’s sarcoma/PNET tumors of the lung are rare22,23 and differ from the present case, which, per se,
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does not fit either into the usual neuroendocrine carcinoma of the lung. Furthermore, in this age-group, poorly differ-entiated neuroendocrine carcinomas are even rarer and, if improperly treated, threaten, in the short term, the patient survival.
The patient in this report was treated with neoadjuvant chemotherapy for Ewing’s sarcoma/PNET according to Euro Ewing 99 study protocol followed by surgery and has no evidence of disease at the time of writing, 15 months after the initial diagnosis. This highlights the importance of achieving the correct diagnosis of these atypical tumors using all clinical, morphological, and ancillary methods available to allow for their correct and timely treatment. It remains to be seen in larger series of uncommon Ewing’s sarcoma/PNET tumors with longer follow-up whether it will be possible to improve patient’s treatment by adding the presence of unusual morphologic and immunohisto-chemical features to the typical molecular signature.
Acknowledgments
The authors thank Dr Saul Suster for the second opinion on the case reported here; Dr Fatima Magalhaes for providing the clini-cal and pathological information from the first diagnosis; and Dr Catarina Eloy for sharing a similar thyroid case with us.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
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