Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
First evidence of treatment efficacy in metastatic carcinoma of the parotid gland with BRD4/NUT translocation Christof Vulsteke, Eveline Lurquin, Maria Debiec-Rychter, Olivier Gheysens, Sandra Nuyts, Joseph Schoenaers, Constantinus Politis, Jeroen Mebis, Esther Hauben & Paul M. Clement To cite this article: Christof Vulsteke, Eveline Lurquin, Maria Debiec-Rychter, Olivier Gheysens, Sandra Nuyts, Joseph Schoenaers, Constantinus Politis, Jeroen Mebis, Esther Hauben & Paul M. Clement (2016) First evidence of treatment efficacy in metastatic carcinoma of the parotid gland with BRD4/NUT translocation, Journal of Chemotherapy, 28:3, 242-246, DOI: 10.1179/1973947815Y.0000000046 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000046
Published online: 14 Jul 2016.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Cornell University Library]
Date: 21 August 2016, At: 09:03
Case Report
First evidence of treatment efficacy in metastatic carcinoma of the parotid gland with BRD4/NUT translocation Christof Vulsteke1,2, Eveline Lurquin3, Maria Debiec-Rychter4, Olivier Gheysens3, Sandra Nuyts5,6, Joseph Schoenaers7, Constantinus Politis7, Jeroen Mebis8, Esther Hauben3, Paul M. Clement2,6 1
Department of Oncology, Integrated Cancer Center, Ghent, Belgium, 2Department of General Medical Oncology, UZ Leuven, Belgium, 3Department of Imaging and Pathology, KU Leuven, Belgium, 4Department of Human Genetics, KU Leuven, Belgium, 5Department of Radiation Oncology, UZ Leuven, Belgium, 6Department of Oncology, KU Leuven, Belgium, 7Department of Oral Health Sciences, KU Leuven, Belgium, 8Department of Medical Oncology, Jessa Ziekenhuis, Hasselt, Belgium Background: Nuclear protein in testis (NUT) midline carcinomas (NMC) are characterized by rearrangements of the gene NUT. In the majority of NMCs, a translocation t(15;19), resulting in a BRD4/NUT fusion gene, is present. Nuclear protein in testis midline carcinomas is a rare, but probably underdiagnosed entity due to misdiagnosis. Most cases have been reported in the mediastinum and upper aerodigestive tract. The clinical course of a NMC is extremely aggressive, in spite of intensive chemotherapy and radiotherapy, with an average survival v1 year. Methods and results: A 32-year-old man presented with a pre-auricular swelling on the left side. After partial parotidectomy, the diagnosis of a NMC was made based on the presence of t(15;19)(q14;p13.1) and BRD4/NUT fusion gene demonstrated by fluorescence in situ hybridization (FISH). During postoperative radiotherapy, the patient developed bone metastases for which chemotherapy consisting of cisplatine, doxorubicine and ifosfamide (PAI) was initiated with remarkable clinical and radiological improvement. Nevertheless, the response was not durable. Conclusion: This case illustrates that responses to chemotherapy in the palliative treatment of a t(15;19)translocated salivary gland carcinoma are possible but not durable. Keywords: Nut midline carcinoma, Chemotherapy, BRD4/NUT fusion gene
Introduction Nuclear protein in testis (NUT) germline carcinomas (NMCs), genetically defined by rearrangements of NUT, first came to attention after two reports in 1999 of a 12-year-old child with a tumour of the epiglottis with abrupt fatal outcome despite aggressive multimodality treatment.1 The translocation that was found in this young girl’s tumour, t(15;19)(q13;13.1), led to a review of the literature and revealed the existence of three isolated case reports of paediatric thymic carcinomas with associated t(15;19) translocations, all aggressive and rapidly lethal. At present, several NMCs have been described, of which the majority occurs in the aero-digestive tract, particularly the sinonasal region and mediastinum.2 Exceptional cases have arisen in bone,3 bladder,2 abdominal retroperito-
neum, pancreas and salivary glands.4,5 The concept that NMCs are confined to children and young adults has been abandoned.1,6,7 Despite aggressive chemotherapy and radiation treatment, the average survival remains v1 year However, one report describes a BRD4/NUT-positive undifferentiated tumour of the iliac bone in complete remission for more than 13 years after combined modality therapy according to the Scandinavian Sarcoma Group (SSG) IX protocol for inoperable Ewing Sarcoma.3 This protocol featured a programme of four chemotherapy cycles, each consisting of two courses of VAI (vincristine, doxorubicin and ifosfamide) alternating with one course of PAI (cisplatin, doxorubicin and ifosfamide) at three weekly intervals.8 During cycles 1 and 2, hyperfractionated accelerated radiotherapy (1.5 Gy twice daily to a total dose of
Correspondence to: Christof Vulsteke, V Department of Oncology, Integrated Cancer Center, Ghent, Belgium. Emails: [email protected]; [email protected]
242
ß 2016 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000046
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60 Gy) was administered to the tumour area. In the metastatic setting, a response to docetaxel was reported,9 but there is no response experience with PAI or VAI. In fact, in a systematic review, chemotherapy has been reported to be inactive in this condition.6 In this report, we describe a remarkable response to PAI chemotherapy in a patient with diffuse bone metastases of a NMC of the parotid gland.
Methods and Results Initial presentation A 32-year-old man presented in 2013 with a rapidly growing pre-auricular tumour on the left side. A magnetic resonance scan revealed a subcutaneous solid mass at the left cheek probably originating from the accessory lobe of the parotid gland, in close contact with the fascia of the masseter muscle and without invasion of adjacent structures. Fine needle aspiration yielded a mixture of necrotic and viable cells, immunohistochemically suggestive for a malignant epithelial tumour (diffuse positivity for prekeratin and negative staining for S100 protein).
Immunohistochemistry A macroscopically total resection was performed (partial parotidectomy) showing an infiltrative nodular mass with a solid growth pattern of undifferentiated cells and with a size of 3.8|2.4|2.2 cm. The tumour cells were monotonous with prominent vesicular nuclei and high mitotic and apoptotic activity. Perineural invasion was present and surgical margins were positive. Immunostaining of the surgical biopsy showed diffuse positivity for prekeratin, a moderate diffuse cytoplasmatic positivity for alfa-SMA, a focal cytoplasmatic expression of synaptophysin and NCAM, and a strong, near 100% nuclear reactivity for p63. Period acid-Schiff (PAS) staining was positive for multiple cytoplasmatic granules disappearing after alfa-amylase pretreatment. Awaiting the results of cytogenetic analysis, the pathologist concluded to a poorly differentiated carcinoma and suggested a myoepithelial carcinoma with neuro-endocrine features (Fig. 1).
Cytogenetic analysis and fluorescence in situ hybridization Cytogenetic analysis was performed on Giemsabanded metaphase spreads obtained from shortterm primary tumour’s tissue culture, using standard procedures. Fluorescence in situ hybridization (FISH) on interphase nuclei on single tumour cells suspension was performed applying custom probes, using bacterial artificial chromosome (BAC) clones, which were chosen according to the USCS genome browser (http://genome.uscs.edu). Bacterial artificial chromosome clones were obtained from PAC-PAC sources of Children’s Hospital of Oakland Research
Nuclear protein in testis midline carcinoma
Institute (CHORI; Oakland, CA, USA; http://bac pac.chori.org). A bring-together FISH probe for BRD4-NUT fusion was constructed by labelling the BAC RP11-207I16 probe (telomeric to NUT/15q14) in spectrum orange (SO) and the BAC RP11602M11 probe (centromeric to BRD4/19p13.1) in spectrum green (SG). DNA isolation, nick translation and hybridization and FISH evaluation were performed as described previously.10 Cytogenetic analysis of tumour metaphases revealed an abnormal karyotype with t(15;19)(q14;p13.1) as the sole anomaly (Fig. 2). Sequentially, a staining for NUT using NUT (C52B1) rabbit monoclonal antibody was performed showing nuclear positivity. Interphase FISH analysis confirmed the presence of the BRD4-NUT fusion in all analysed cells (Fig. 3).
Staging and treatment Postoperative computed tomography of the head and neck area showed a nodule in the surgical region suggestive for residual tumour. After multidisciplinary assessment and a negative staging with computed tomography of the thorax and abdomen, a postoperative irradiation was initiated. A total dose of 66 Gy in 33 fractions of 2 Gy was administrated to the left neck and parotid gland remnant, using intensity modulated radiation therapy. The tumour bed received a total dose of 69.96 Gy in 33 fractions. In the third week of radiotherapy, the patient complained of abdominal pain, which led us to a prescheduled evaluation with computed tomography of the thorax and abdomen now showing diffuse osteolytic lesions in the bone and a pathologic fracture of the sixth rib based on an expansive bone lesion. Pain medication was started and the radiotherapy course was continued as planned. One month after the CT thorax and abdomen, an additional FDG-PET/CT scan was performed showing intense hypermetabolism of the osteolytic bone lesions and a large soft tissue component of the metastatic lesion in the right sixth rib. The patient was reassessed multidisciplinary and a chemotherapy regimen consisting of cisplatin 2 2 30– 40 mg/m , doxorubicin 20 mg/m and ifosfamide 2000 mg/m2 from day 1–3 inches a three weekly schedule (PAI) was proposed, as well as a monthly administration of denosumab. The patient developed febrile neutropaenia, as well as Grade II nausea and anorexia, leading to a therapeutic change to CAP (cyclophosphamide 600 mg/m2, doxorubicin 50 mg/m2 and cisplatinum 50 mg/m2, three weekly schedule on day 1) after two cycles. Besides that, a radiation recall phenomenon was observed (left side mucositis only). Nevertheless, the patient reported a marked improvement of pain and a decreasing need for analgesics. Reevaluation with FDG-PET/CT after two cycles of PAI showed
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Figure 1 a: Shows a nodular solid tumoral mass (right) adjacent to normal parotic parenchyma (left), b: Prominent vesicular nuclei with high mitotic and apoptotic activity, c: Diffuse cytoplasmatic expression for alfa-SMA, d: Weak to moderate, granular staining of the nuclei with the NUT antibody.
showed progressive disease with multiple new bone lesions (and no visceral metastases). A best supportive care setting was started and the patient eventually died four months later.
Discussion
Figure 2 GTG-banded karyotype showing t(15;19)(q14;p13.1) translocation (arrows).
a
sole
a very good metabolic response with only minor residual activity of some bone lesions and a complete metabolic response of other lesions. In addition, a marked regression of the expansive soft tissue lesion of the sixth rib was observed (Fig. 4). This response was maintained after four cycles of subsequent CAP. Two months later, however, the patient complained again of evolving bone pain. Reevaluation with bone scan and computed tomography of the thorax and abdomen
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Nuclear protein in testis (NUT) midline carcinomas is most likely more common than currently thought. Since histologic features are that of a poorly differentiated carcinoma, NMC is often misdiagnosed as a squamous cell carcinoma, Ewing sarcoma, leukaemia, sinonasal undifferentiated carcinoma or pancreatoblastoma. The corner stone of the diagnosis is made by karyotyping or FISH, though cryptic NUT rearrangements can be missed.11 Therefore, most sensitive approach is to both perform NUT IHC and FISH. However, FISH assay is often not available in pathology or molecular laboratories. Staining with the NUT antibody by immunohistochemistry can be used instead with a good sensitivity and high specificity. Knowing when to test for NMC is the biggest challenge and it has been proposed that a useful guideline is to consider NMC in the differential diagnosis of every midline poorly differentiated carcinoma in a never-smoker and in the absence of
Vulsteke et al.
Nuclear protein in testis midline carcinoma
Figure 3 Interphase dual-colour FISH analysis by co-hybridization of the differentially labelled BAC probes RP11-207I16-SG (green) and RP11-602M11-SO (red) revealed the BRD4-NUT fusion, as evidenced by the presence of juxtaposed green and red hybridization signals (arrows) in tumour’s nuclei.
Figure 4 A 32-year-old man with NUT midline carcinoma. Baseline FDG-PET/CT (A –D) and FDG-PET/CT after 2 cycles of chemotherapy (E – H). Maximum intensity projection images (A, E). transaxial PET (B, F), CT (C, G) and fused PET-CT (D, H) images. Baseline FDG-PET/CT reveals multiple bone lesions with moderate to strong uptake and an expansive soft tissue mass in the sixth rib (arrow). Reevaluation after chemotherapy showed a very good response with minimal residual activity at the sixth rib (arrow) and a nearly complete metabolic response of all other skeletal lesions.
human papillomavirus or Epstein-Barr virus in the tumours1,10 In our patient, the diagnosis was made because fresh material from the majority of solid tumours is routinely sent to the cytogenetic lab for karyotyping. However, this is not considered a feasible practice for most institutions. An alternative is to include the NUT antigen in the immunohistochemical panel for poorly and undifferentiated tumours. It has to be taken in account that these carcinomas show squamous and even myoepithelial differentiation on immunohistochemistry. The aggressive expansion of the tumour
and the development of diffuse bone metastasis two months after staging are compatible with the aggressive behaviour and poor prognosis of the reported NMC cases in literature. Local control of the primary tumour is possible, as demonstrated by retrospective series that is also suggestive of an improved progression-free and overall survival after complete resection and radiation. In the metastatic setting, no chemotherapeutic regimen (including platinum, alkylating agents, taxanes or anthracycline-based regimens) has shown durable responses.6 BRD4-NUT is
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believed to be responsible for blocking differentiation and maintaining the cells in a perpetual state of proliferation in NMC. Two biological therapies that target this oncogenic mechanism are described and show activity in vitro and in vivo. The first is a bromodomain inhibitor that is currently available for adults with NMC through a phase I/II clinical trial with GSK525762 and the second is the histone-deacetylase inhibitor vorinostat that demonstrated a clinical response in a paediatric patient.12 However, the treatment had to be interrupted because of toxicity and immediate recurrence was observed. Our case report is of particular clinical interest. First, it confirms multiple reports showing a lack of durable response to chemotherapy. In fact, disease typically progresses within 2 or 3 months of chemotherapy leading to fatal outcome (reported median overall survival of 6.7 months).6 Second, the location of the primary tumour is exceptional. In the literature, only one case report has been described of a NMC in the parotid gland. This 15year old patient was treated with adjuvant radiotherapy concomitant with chemotherapy.4 In conclusion, we believe that awareness of the possibility of a t(15;19)-translocation in a poorly differentiated tumour in the parotid gland or in a midline tumour is important, because of its association with poor survival. Or case report confirms that responses to chemotherapy are not durable. Therefore, we hope for improved feasibility and efficacy of new targetted therapies.
Disclaimer Statements Contributors Christof Vulsteke was involved in data collection, supervised chemotherapy and wrote the manuscript. Eveline Lurquin provided the figures and critically revised the manuscript. Maria Debiec-Rychter performed FISH analysis, provided the figures and critically revised the manuscript. Olivier Gheysens provided the figures and critically revised the manuscript. Sandra Nuyts performed radiotherapy and critically revised the manuscript. Joseph Schoenaers performed the surgery and critically revised the manuscript. Constantinus Politis performed the surgery and critically revised the manuscript. Jeroen Mebis
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supervised chemotherapy and critically revised the manuscript. Esther Hauben performed immunostaining and critically revised the manuscript. Paul M. Clement supervised chemotherapy and critically revised the manuscript. Funding No funding or financial support. Conflicts of interest No Conflict of Interest for all authors. Ethics approval The collection of patient data was approved by ethics committee of UZ Leuven.
References 1 French CA. NUT midline carcinoma. Cancer Genet Cytogenet. 2010;203(1):16–20. 2 Stelow EB, Bellizzi AM, Taneja K, Mills SE, Legallo RD, Kutok JL, et al. NUT rearrangement in undifferentiated carcinomas of the upper aerodigestive tract. Am J Surg Pathol. 2008;32(6):828–834. 3 Mertens F, Wiebe T, Adlercreutz C, Mandahl N, French CA. Successful treatment of a child with t(15;19)-positive tumor. Pediatr Blood Cancer. 2007;49(7):1015–1057. 4 den Bakker MA, Beverloo BH, van den Heuvel-Eibrink MM, Meeuwis CA, Tan LM, Johnson LA, et al. NUT midline carcinoma of the parotid gland with mesenchymal differentiation. Am J Surg Pathol. 2009;33(8):1253–1258. 5 Ziai J, French CA, Zambrano E. NUT gene rearrangement in a poorly-differentiated carcinoma of the submandibular gland. Head Neck Pathol. 2010;4(2):163–168. 6 Bauer DE, Mitchell CM, Strait KM, Lathan CS, Stelow EB, Luer SC, et al. Clinicopathologic features and long-term outcomes of NUT midline carcinoma. Clin Cancer Res. 2012;18(20):5773–5779. 7 French CA, Kutok JL, Faquin WC, Toretsky JA, Antonescu CR, Griffin CA, et al. Midline carcinoma of children and young adults with NUT rearrangement. J Clin Oncol. 2004;22(20):4135–4139. 8 Elomaa I, Blomqvist CP, Saeter G, Akerman M, Stenwig E, Wiebe T, et al. Five-year results in Ewing’s sarcoma. The Scandinavian sarcoma group experience with the SSG IX protocol. Eur J Cancer. 2000;36(7):875–880. 9 Engleson J, Soller M, Panagopoulos I, Dahien A, Dictor M, Jerkeman M. Midline carcinoma with t(15;19) and BRD4-NUT fusion oncogene in a 30-year-old female with response to docetaxel and radiotherapy. BMC Cancer. 2006;6:69. 10 Debiec-Rychter M, Croes R, De Vos R, Marynen P, Roskams T, Hagemeijer A, et al. Complex genomic rearrangement of ALK loci associated with integrated human Epstein-Barr virus in a post-transplant myogenic liver tumor. Am J Pathol. 2003;163(3):913–922. 11 Haack H, Johnson LA, Fry CJ, Crosby K, Polakiewicz RD, Stelow EB, et al. Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody. Am J Surg Pathol. 2009;33(7):984–991. 12 Schwartz BE, Hofer MD, Lemieux ME, Bauer DE, Cameron MJ, West NH, et al. Differentiation of NUT midline carcinoma by epigenomic reprogramming. Cancer Res. 2011;71(7): 2686–2696.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
First evidence of treatment efficacy in metastatic carcinoma of the parotid gland with BRD4/NUT translocation Christof Vulsteke, Eveline Lurquin, Maria Debiec-Rychter, Olivier Gheysens, Sandra Nuyts, Joseph Schoenaers, Constantinus Politis, Jeroen Mebis, Esther Hauben & Paul M. Clement To cite this article: Christof Vulsteke, Eveline Lurquin, Maria Debiec-Rychter, Olivier Gheysens, Sandra Nuyts, Joseph Schoenaers, Constantinus Politis, Jeroen Mebis, Esther Hauben & Paul M. Clement (2016) First evidence of treatment efficacy in metastatic carcinoma of the parotid gland with BRD4/NUT translocation, Journal of Chemotherapy, 28:3, 242-246, DOI: 10.1179/1973947815Y.0000000046 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000046
Published online: 14 Jul 2016.
Submit your article to this journal
Article views: 16
View related articles
View Crossmark data
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Cornell University Library]
Date: 21 August 2016, At: 09:03
Case Report
First evidence of treatment efficacy in metastatic carcinoma of the parotid gland with BRD4/NUT translocation Christof Vulsteke1,2, Eveline Lurquin3, Maria Debiec-Rychter4, Olivier Gheysens3, Sandra Nuyts5,6, Joseph Schoenaers7, Constantinus Politis7, Jeroen Mebis8, Esther Hauben3, Paul M. Clement2,6 1
Department of Oncology, Integrated Cancer Center, Ghent, Belgium, 2Department of General Medical Oncology, UZ Leuven, Belgium, 3Department of Imaging and Pathology, KU Leuven, Belgium, 4Department of Human Genetics, KU Leuven, Belgium, 5Department of Radiation Oncology, UZ Leuven, Belgium, 6Department of Oncology, KU Leuven, Belgium, 7Department of Oral Health Sciences, KU Leuven, Belgium, 8Department of Medical Oncology, Jessa Ziekenhuis, Hasselt, Belgium Background: Nuclear protein in testis (NUT) midline carcinomas (NMC) are characterized by rearrangements of the gene NUT. In the majority of NMCs, a translocation t(15;19), resulting in a BRD4/NUT fusion gene, is present. Nuclear protein in testis midline carcinomas is a rare, but probably underdiagnosed entity due to misdiagnosis. Most cases have been reported in the mediastinum and upper aerodigestive tract. The clinical course of a NMC is extremely aggressive, in spite of intensive chemotherapy and radiotherapy, with an average survival v1 year. Methods and results: A 32-year-old man presented with a pre-auricular swelling on the left side. After partial parotidectomy, the diagnosis of a NMC was made based on the presence of t(15;19)(q14;p13.1) and BRD4/NUT fusion gene demonstrated by fluorescence in situ hybridization (FISH). During postoperative radiotherapy, the patient developed bone metastases for which chemotherapy consisting of cisplatine, doxorubicine and ifosfamide (PAI) was initiated with remarkable clinical and radiological improvement. Nevertheless, the response was not durable. Conclusion: This case illustrates that responses to chemotherapy in the palliative treatment of a t(15;19)translocated salivary gland carcinoma are possible but not durable. Keywords: Nut midline carcinoma, Chemotherapy, BRD4/NUT fusion gene
Introduction Nuclear protein in testis (NUT) germline carcinomas (NMCs), genetically defined by rearrangements of NUT, first came to attention after two reports in 1999 of a 12-year-old child with a tumour of the epiglottis with abrupt fatal outcome despite aggressive multimodality treatment.1 The translocation that was found in this young girl’s tumour, t(15;19)(q13;13.1), led to a review of the literature and revealed the existence of three isolated case reports of paediatric thymic carcinomas with associated t(15;19) translocations, all aggressive and rapidly lethal. At present, several NMCs have been described, of which the majority occurs in the aero-digestive tract, particularly the sinonasal region and mediastinum.2 Exceptional cases have arisen in bone,3 bladder,2 abdominal retroperito-
neum, pancreas and salivary glands.4,5 The concept that NMCs are confined to children and young adults has been abandoned.1,6,7 Despite aggressive chemotherapy and radiation treatment, the average survival remains v1 year However, one report describes a BRD4/NUT-positive undifferentiated tumour of the iliac bone in complete remission for more than 13 years after combined modality therapy according to the Scandinavian Sarcoma Group (SSG) IX protocol for inoperable Ewing Sarcoma.3 This protocol featured a programme of four chemotherapy cycles, each consisting of two courses of VAI (vincristine, doxorubicin and ifosfamide) alternating with one course of PAI (cisplatin, doxorubicin and ifosfamide) at three weekly intervals.8 During cycles 1 and 2, hyperfractionated accelerated radiotherapy (1.5 Gy twice daily to a total dose of
Correspondence to: Christof Vulsteke, V Department of Oncology, Integrated Cancer Center, Ghent, Belgium. Emails: [email protected]; [email protected]
242
ß 2016 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000046
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60 Gy) was administered to the tumour area. In the metastatic setting, a response to docetaxel was reported,9 but there is no response experience with PAI or VAI. In fact, in a systematic review, chemotherapy has been reported to be inactive in this condition.6 In this report, we describe a remarkable response to PAI chemotherapy in a patient with diffuse bone metastases of a NMC of the parotid gland.
Methods and Results Initial presentation A 32-year-old man presented in 2013 with a rapidly growing pre-auricular tumour on the left side. A magnetic resonance scan revealed a subcutaneous solid mass at the left cheek probably originating from the accessory lobe of the parotid gland, in close contact with the fascia of the masseter muscle and without invasion of adjacent structures. Fine needle aspiration yielded a mixture of necrotic and viable cells, immunohistochemically suggestive for a malignant epithelial tumour (diffuse positivity for prekeratin and negative staining for S100 protein).
Immunohistochemistry A macroscopically total resection was performed (partial parotidectomy) showing an infiltrative nodular mass with a solid growth pattern of undifferentiated cells and with a size of 3.8|2.4|2.2 cm. The tumour cells were monotonous with prominent vesicular nuclei and high mitotic and apoptotic activity. Perineural invasion was present and surgical margins were positive. Immunostaining of the surgical biopsy showed diffuse positivity for prekeratin, a moderate diffuse cytoplasmatic positivity for alfa-SMA, a focal cytoplasmatic expression of synaptophysin and NCAM, and a strong, near 100% nuclear reactivity for p63. Period acid-Schiff (PAS) staining was positive for multiple cytoplasmatic granules disappearing after alfa-amylase pretreatment. Awaiting the results of cytogenetic analysis, the pathologist concluded to a poorly differentiated carcinoma and suggested a myoepithelial carcinoma with neuro-endocrine features (Fig. 1).
Cytogenetic analysis and fluorescence in situ hybridization Cytogenetic analysis was performed on Giemsabanded metaphase spreads obtained from shortterm primary tumour’s tissue culture, using standard procedures. Fluorescence in situ hybridization (FISH) on interphase nuclei on single tumour cells suspension was performed applying custom probes, using bacterial artificial chromosome (BAC) clones, which were chosen according to the USCS genome browser (http://genome.uscs.edu). Bacterial artificial chromosome clones were obtained from PAC-PAC sources of Children’s Hospital of Oakland Research
Nuclear protein in testis midline carcinoma
Institute (CHORI; Oakland, CA, USA; http://bac pac.chori.org). A bring-together FISH probe for BRD4-NUT fusion was constructed by labelling the BAC RP11-207I16 probe (telomeric to NUT/15q14) in spectrum orange (SO) and the BAC RP11602M11 probe (centromeric to BRD4/19p13.1) in spectrum green (SG). DNA isolation, nick translation and hybridization and FISH evaluation were performed as described previously.10 Cytogenetic analysis of tumour metaphases revealed an abnormal karyotype with t(15;19)(q14;p13.1) as the sole anomaly (Fig. 2). Sequentially, a staining for NUT using NUT (C52B1) rabbit monoclonal antibody was performed showing nuclear positivity. Interphase FISH analysis confirmed the presence of the BRD4-NUT fusion in all analysed cells (Fig. 3).
Staging and treatment Postoperative computed tomography of the head and neck area showed a nodule in the surgical region suggestive for residual tumour. After multidisciplinary assessment and a negative staging with computed tomography of the thorax and abdomen, a postoperative irradiation was initiated. A total dose of 66 Gy in 33 fractions of 2 Gy was administrated to the left neck and parotid gland remnant, using intensity modulated radiation therapy. The tumour bed received a total dose of 69.96 Gy in 33 fractions. In the third week of radiotherapy, the patient complained of abdominal pain, which led us to a prescheduled evaluation with computed tomography of the thorax and abdomen now showing diffuse osteolytic lesions in the bone and a pathologic fracture of the sixth rib based on an expansive bone lesion. Pain medication was started and the radiotherapy course was continued as planned. One month after the CT thorax and abdomen, an additional FDG-PET/CT scan was performed showing intense hypermetabolism of the osteolytic bone lesions and a large soft tissue component of the metastatic lesion in the right sixth rib. The patient was reassessed multidisciplinary and a chemotherapy regimen consisting of cisplatin 2 2 30– 40 mg/m , doxorubicin 20 mg/m and ifosfamide 2000 mg/m2 from day 1–3 inches a three weekly schedule (PAI) was proposed, as well as a monthly administration of denosumab. The patient developed febrile neutropaenia, as well as Grade II nausea and anorexia, leading to a therapeutic change to CAP (cyclophosphamide 600 mg/m2, doxorubicin 50 mg/m2 and cisplatinum 50 mg/m2, three weekly schedule on day 1) after two cycles. Besides that, a radiation recall phenomenon was observed (left side mucositis only). Nevertheless, the patient reported a marked improvement of pain and a decreasing need for analgesics. Reevaluation with FDG-PET/CT after two cycles of PAI showed
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Figure 1 a: Shows a nodular solid tumoral mass (right) adjacent to normal parotic parenchyma (left), b: Prominent vesicular nuclei with high mitotic and apoptotic activity, c: Diffuse cytoplasmatic expression for alfa-SMA, d: Weak to moderate, granular staining of the nuclei with the NUT antibody.
showed progressive disease with multiple new bone lesions (and no visceral metastases). A best supportive care setting was started and the patient eventually died four months later.
Discussion
Figure 2 GTG-banded karyotype showing t(15;19)(q14;p13.1) translocation (arrows).
a
sole
a very good metabolic response with only minor residual activity of some bone lesions and a complete metabolic response of other lesions. In addition, a marked regression of the expansive soft tissue lesion of the sixth rib was observed (Fig. 4). This response was maintained after four cycles of subsequent CAP. Two months later, however, the patient complained again of evolving bone pain. Reevaluation with bone scan and computed tomography of the thorax and abdomen
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Nuclear protein in testis (NUT) midline carcinomas is most likely more common than currently thought. Since histologic features are that of a poorly differentiated carcinoma, NMC is often misdiagnosed as a squamous cell carcinoma, Ewing sarcoma, leukaemia, sinonasal undifferentiated carcinoma or pancreatoblastoma. The corner stone of the diagnosis is made by karyotyping or FISH, though cryptic NUT rearrangements can be missed.11 Therefore, most sensitive approach is to both perform NUT IHC and FISH. However, FISH assay is often not available in pathology or molecular laboratories. Staining with the NUT antibody by immunohistochemistry can be used instead with a good sensitivity and high specificity. Knowing when to test for NMC is the biggest challenge and it has been proposed that a useful guideline is to consider NMC in the differential diagnosis of every midline poorly differentiated carcinoma in a never-smoker and in the absence of
Vulsteke et al.
Nuclear protein in testis midline carcinoma
Figure 3 Interphase dual-colour FISH analysis by co-hybridization of the differentially labelled BAC probes RP11-207I16-SG (green) and RP11-602M11-SO (red) revealed the BRD4-NUT fusion, as evidenced by the presence of juxtaposed green and red hybridization signals (arrows) in tumour’s nuclei.
Figure 4 A 32-year-old man with NUT midline carcinoma. Baseline FDG-PET/CT (A –D) and FDG-PET/CT after 2 cycles of chemotherapy (E – H). Maximum intensity projection images (A, E). transaxial PET (B, F), CT (C, G) and fused PET-CT (D, H) images. Baseline FDG-PET/CT reveals multiple bone lesions with moderate to strong uptake and an expansive soft tissue mass in the sixth rib (arrow). Reevaluation after chemotherapy showed a very good response with minimal residual activity at the sixth rib (arrow) and a nearly complete metabolic response of all other skeletal lesions.
human papillomavirus or Epstein-Barr virus in the tumours1,10 In our patient, the diagnosis was made because fresh material from the majority of solid tumours is routinely sent to the cytogenetic lab for karyotyping. However, this is not considered a feasible practice for most institutions. An alternative is to include the NUT antigen in the immunohistochemical panel for poorly and undifferentiated tumours. It has to be taken in account that these carcinomas show squamous and even myoepithelial differentiation on immunohistochemistry. The aggressive expansion of the tumour
and the development of diffuse bone metastasis two months after staging are compatible with the aggressive behaviour and poor prognosis of the reported NMC cases in literature. Local control of the primary tumour is possible, as demonstrated by retrospective series that is also suggestive of an improved progression-free and overall survival after complete resection and radiation. In the metastatic setting, no chemotherapeutic regimen (including platinum, alkylating agents, taxanes or anthracycline-based regimens) has shown durable responses.6 BRD4-NUT is
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believed to be responsible for blocking differentiation and maintaining the cells in a perpetual state of proliferation in NMC. Two biological therapies that target this oncogenic mechanism are described and show activity in vitro and in vivo. The first is a bromodomain inhibitor that is currently available for adults with NMC through a phase I/II clinical trial with GSK525762 and the second is the histone-deacetylase inhibitor vorinostat that demonstrated a clinical response in a paediatric patient.12 However, the treatment had to be interrupted because of toxicity and immediate recurrence was observed. Our case report is of particular clinical interest. First, it confirms multiple reports showing a lack of durable response to chemotherapy. In fact, disease typically progresses within 2 or 3 months of chemotherapy leading to fatal outcome (reported median overall survival of 6.7 months).6 Second, the location of the primary tumour is exceptional. In the literature, only one case report has been described of a NMC in the parotid gland. This 15year old patient was treated with adjuvant radiotherapy concomitant with chemotherapy.4 In conclusion, we believe that awareness of the possibility of a t(15;19)-translocation in a poorly differentiated tumour in the parotid gland or in a midline tumour is important, because of its association with poor survival. Or case report confirms that responses to chemotherapy are not durable. Therefore, we hope for improved feasibility and efficacy of new targetted therapies.
Disclaimer Statements Contributors Christof Vulsteke was involved in data collection, supervised chemotherapy and wrote the manuscript. Eveline Lurquin provided the figures and critically revised the manuscript. Maria Debiec-Rychter performed FISH analysis, provided the figures and critically revised the manuscript. Olivier Gheysens provided the figures and critically revised the manuscript. Sandra Nuyts performed radiotherapy and critically revised the manuscript. Joseph Schoenaers performed the surgery and critically revised the manuscript. Constantinus Politis performed the surgery and critically revised the manuscript. Jeroen Mebis
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supervised chemotherapy and critically revised the manuscript. Esther Hauben performed immunostaining and critically revised the manuscript. Paul M. Clement supervised chemotherapy and critically revised the manuscript. Funding No funding or financial support. Conflicts of interest No Conflict of Interest for all authors. Ethics approval The collection of patient data was approved by ethics committee of UZ Leuven.
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