RESEARCH ARTICLE
Expression of P16 in NUT Carcinomas With No Association With Human Papillomavirus (HPV) Paulo Guilherme de Oliveira Salles, MD, PhD,* Rafael de Deus Moura, MD,w Letı´cia M. Menezes, MD,* and Carlos E. Bacchi, MD, PhDw
Abstract: NUT carcinoma (NC) is a rare malignant neoplasm usually located in the midline, including the upper aerodigestive tract. NC is an aggressive and highly lethal type of carcinoma. It is defined by the rearrangement of the nuclear protein in the testis (NUT) gene on chromosome 15q14. In most cases, the NUT is involved in a balanced translocation with the BRD4 gene on chromosome 19p13.1, an event that creates a BRD4-NUT fusion gene. The relationship between the human papillomavirus (HPV), p16, and upper aerodigestive tract cancer has been long postulated. In this study, we evaluated the relationship of the p16 expression in 4 cases of NCs and its eventual association with HPV. All 4 cases presented typical histopathologic findings with nuclear positivity of the NUT protein and strong expression for p16. None of these cases, however, showed an association with HPV evaluated by polymerase chain reaction. Despite the expression of p16, this negative result for HPV indicates that HPV infection probably does not play a role in the pathogenesis of NC. Key Words: NUT carcinoma, p16, HPV, immunohistochemistry (Appl Immunohistochem Mol Morphol 2014;22:262–265)
N
UT carcinoma (NC) is a rare, aggressive, and highly lethal cancer that occurs in children and adults of all ages. Most NCs are present in the midline of the upper aerodigestive tract or in the mediastinum as poorly differentiated carcinomas with variable degrees of squamous differentiation. The lung is sometimes involved when tumors are large in size, possibly because of secondary extension; there are only a few reports of primary tumors arising in the lung. Some unusual cases have presented below the diaphragm, including tumors that presented in the posterior bladder and the bones of the pelvis. No cases outside the midline axis have been reported. NC is defined by the rearrangement of the nuclear protein in the testis (NUT) gene on chromosome 15q14. In most cases, NUT is involved in a balanced translocation with the BRD4 gene on chromosome 19p13.1, an event that creReceived for publication May 11, 2013; accepted July 12, 2013. From the *Instituto Mario Penna and Biocor Instituto, Belo Horizonte, MG; and wConsultoria em Patologia, Botucatu, SP, Brazil. The authors declare no conflict of interest. Reprints: Carlos E. Bacchi, MD, PhD, Consultoria em Patologia, Rua Major Leoˆnidas Cardoso 739, Botucatu, SP 18602-010, Brazil (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
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ates a BRD4-NUT fusion gene. Variant rearrangements, some involving the BRD3 gene, occur in the remaining cases. The effect of the NUT protein is not clear; however, BRD4 has a role in transcriptional activation and segregation of the human papillomavirus (HPV) during mitosis. Owing to its rarity and lack of characteristic histologic features, most cases of NC currently go unrecognized, and immunohistochemical analysis using the NUT antibody followed by FISH with probes for the 15q14 NUT break-point may be extremely helpful in the identification of these tumors.1,2 The cyclin-dependent kinase inhibitor 2A (CDKN2A, p16Ink4A), also known as multiple tumor suppressor 1, is a tumor-suppressor protein that plays an important role in regulating the cell cycle, and mutations in p16 increase the risk for developing a variety of cancers. It decelerates the progression of the cell cycle by inactivating the cyclin-dependent kinase that phosphorylates the retinoblastoma protein (pRB). The progression of cells from the G1 phase to the S phase is blocked by protein p16, which acts to disrupt the cyclin D1-CDK4/6 complex. The most critical point in cell-cycle regulation is the G1 checkpoint and it is at this checkpoint that the complex cyclin D1 interactions occur. They determine whether the cell cycle goes back into a quiescent state (G0) or enters the S phase, where cells are destined to divide. When cells enter the S phase, they divide uncontrollably, which leads to cancer.3 A relationship between HPV, p16, and upper aerodigestive tract cancer has long been postulated. HPV-associated squamous cell carcinomas represent a distinct disease entity from carcinogen-associated squamous cell carcinomas. HPV oncoproteins lead to mucosal cell transformation through well-defined mechanisms. The HPV E6 and E7 oncoproteins inactivate the p53 and pRB tumor suppressors, respectively, leading to hyperproliferation and genomic instability. RB inhibits the progression of cells into the S phase and is regulated by cyclin D1 by phosphorylation. Progressive and prolonged phosphorylation of the pRB leads to its inactivation and reduction of its growth suppression activity. This inactivation is mediated by the release of E2F-like transcription factors from RB, which allows the activation of CDK and transcriptional activation of target promoters. The CDKN2A gene product, the p16INK4A protein, inhibits CDK4 and CDK6, which phosphorylate the pRB. A reciprocal relationship between p16INK4A and RB expression
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has been observed. Although the virus is detected in a number of upper aerodigestive tract subsites, studies demonstrate improved outcomes in HPV-associated carcinoma of the oropharynx only. The cell-cycle regulatory protein p16 is upregulated by biologically active HPV and serves as a biomarker of improved response to therapy.3 Our goal was to study the relationship that appears as not yet been described between p16 expression and NCs and the eventual association between this unusual type of carcinoma and HPV.
MATERIALS AND METHODS Case Selection and Pathologic Diagnosis The present series includes 4 cases of NUT midline carcinoma. All cases were retrieved from the archives of Consultoria em Patologia, a large anatomic pathology reference laboratory located in Botucatu, Sa˜o Paulo, Brazil, during the period of April 2010 to January 2012.
Immunohistochemistry Tissues from all cases were formalin fixed and paraffin embedded. The hematoxylin and eosin–stained slides were reviewed for routine morphology. Immunohistochemical analysis was performed using the following panel of antibodies: cytokeratin (clone AE1/AE3, 1:900; Dako); P63 (clone 4A4, 1:4000; Neomarkers); NUT (clone C5B1, 1:100; Cell Signaling); and p16INK (CINtec, 1:200). Novolink polymer (Novocastra, Newcastle upon Tyne, UK) was used as the detection system, and diaminobenzidine was used as the chromogen.
Polymerase Chain Reaction (PCR) for HPV DNA Extraction and Quality Control The hematoxylin and eosin-stained sections of the specimens underwent microdissection before DNA extraction from the formalin-fixed paraffin-embedded tissues. Selected areas and cervical specimens were submitted directly to tissue protein digestion with 100 mg/mL proteinase K for 12 to 15 hours at 551C. DNA was extracted using the saline method. The nucleic-acid purity was determined by a 260/280 nm optical density measurement using a spectrophotometer. A qualitative size range PCR assay was performed to determine the DNA quality of the formalin fixed paraffin embedded tissue and cytologic cervical samples. The assay consisted of 5 primer sets that amplified products of 100, 200, 300, 400, and up to 600 base pairs, and it has previously been used successfully.4
HPV Detection HPV infection was first evaluated by PCR with consensus primers GP5+ and GP6+, which give an amplicon of 138 to 150 base pairs and allow the detection of a broad spectrum of distinct HPV genotypes.5 In addition to undergoing a generic detection for any HPV subtype, the positive samples were submitted to multiplex PCR for discrimination among infections by the main viral genotypes, either of a low risk (HPV 6 and 11) or high risk for cellular transformation (HPV 16, 18, and r
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33).6,7 All PCR reactions were accomplished with positive and negative controls. The amplified DNA was electrophoresed on 7% polyacrylamide gel. The positive and negative results for the presence of viral DNA obtained through PCR were in agreement with the cytologic and histologic diagnoses.
RESULTS Table 1 summarizes the clinical, immunohistochemical, and molecular features of the NUT midline carcinoma cases. The 4 cases analyzed included 3 men and 1 woman with a mean median age of 31 years (range, 29 to 56 y). Case 1 presented with a nasopharyngeal and ethmoidal sinus tumor with invasion of the orbit. Case 2 presented with a mediastinal mass. In case 3, the patient had multiple nodules in the thorax, central nervous system, parotid gland, lungs, liver, breasts, and adrenal gland. In case 4, the patient had a left lower-lobe lung tumor with multiple cervical and mediastinal lymphadenopathies.
Histologic and Immunohistologic Findings Histologically, all 4 cases were high-grade carcinomas (Fig. 1A), with case 1 showing more overt squamous differentiation and case 4 exhibiting focal areas of keratinization. Cases 2 and 3 were composed of sheets of cohesive undifferentiated epithelioid cells exhibiting vesicular nuclei and prominent nucleoli. Areas of coagulative necrosis, mitotic figures, and apoptotic bodies were frequent in all cases. Glandular differentiation was absent. All 4 cases were diffusely positive for cytokeratin and NUT (Fig. 1B) and at least focally positive for p63. p16 was expressed in all cases in at least 50% of the tumor cells (Fig. 1C). All 4 cases were negative for HPV by PCR.
DISCUSSION NUT midline carcinoma is a rare type of malignant epithelial tumor defined by its typical rearrangement on chromosome 15q14 of the gene encoding the nuclear protein of the testis (NUT).2 NUT expression is normally confined to the germ cells of the testis and ovary and has not been detected in human tumors other than NC.8 The characteristics of NC were first described in 2004,9 and the largest cohort of patients with NC studied to date included 63 patients. NC typically arises in the midline head and neck structures or in the thorax and shows an aggressive behavior with early regional invasion and distant metastasis.10 Initially, it was thought to be a childhood cancer most likely because of a selection bias, as cytogenetic analysis and karyotypes are more often obtained for children’s tumors. More recent studies have shown that NC can affect people of all ages with no predilection for either sex.11 In contrast to most carcinomas, which commonly have complex chromosomal abnormalities, NC typically has simple karyotypes, and in some cases the only aberration is the chromosomal rearrangement involving www.appliedimmunohist.com |
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TABLE 1. Summary of the Clinical, Immunohistochemical, and Molecular Features of the 4 Cases of NUT Midline Carcinoma Age (y) Sex Anatomic location
Cytokeratin p63 NUT p16 HPV-PCR
Case 1
Case 2
Case 3
Case 4
30 Male Nasopharynx/ ethmoidal sinus
29 Male Mediastinum
56 Male Lung and lymph nodes
+ + + +
+ + + +
34 Female Thorax, central nervous system, parotid gland, lungs, liver, breasts, and adrenal + + + +
+ F + +
F indicates focally positive (< 50%); HPV, human papillomavirus; PCR, polymerase chain reaction.
NUT. Those simple karyotypes are typically more present in leukemias, lymphomas, and sarcomas. Considering the pathogenesis of these tumor types, it has been hypothesized that NUT fusion proteins may initiate malignant transformation within epithelial cell precursors and require relatively few collaborative mutations or epigenetic changes to produce NC.12 Morphologically, NCs are indistinguishable from other poorly differentiated carcinomas. Histologically, they are composed of sheets of undifferentiated cells with scant amphophilic or eosinophilic cytoplasm. Occasional cases may have more nested malignant cells within a desmoplastic stroma. An abrupt squamous differentiation is frequently observed with maturing squamous cells and extracellular keratin formation. The cell nuclei have irregular contours, but they tend to have a uniform size with prominent nucleoli and fine vesicular chromatin.2 NC has recently become diagnosable in most cases by immunohistochemical staining with an anti-NUT monoclonal antibody. Improvements in diagnostic methods have led to increased recognition of NC, but its true prevalence remains unknown.13 Few retrospective studies have been conducted concerning the epidemiology of NC. In a study conducted at The Johns Hopkins Hospital, the archival surgical pathology files were searched for all cases of primary sinonasal carcinomas diagnosed from 1995 to 2011; of the 151 cases, only 3 (2%) were NUT positive.14 Evans and colleagues examined the NUT expression in 114 cases of poorly differentiated carcinomas or unclassified
mediastinal malignancies. Four of their cases (3.5%) showed positivity for the NUT expression. Therefore, NC should be considered in the differential diagnosis of any poorly differentiated epithelioid mediastinal tumor, regardless of age.15 Three of 4 of our cases were immunoreactive to p63 antibodies—consistent with squamous differentiation—and all of them were positive for cytokeratin. Interestingly, all 4 cases were also immunoreactive to p16, and despite the presence of p16 expression, none of these tumors showed evidence of HPV infection measured by PCR. p16 is a protein involved in the negative regulation of cell proliferation and is therefore considered a tumor suppressor.3 According to Gonzalez and Serrano,16 close to 50% of all human cancers show p16 inactivation ranging from 25% to 70%. These include head and neck, esophageal, biliary tract, liver, lung, and breast carcinomas. It is well known that p16 contributes to the regulation of cell-cycle progression by inhibiting the S phase. The expression of p16 maintains the Rb family members in a hypophosphorylated state, which promotes binding to E2F1 and leads to G1 cell-cycle arrest.3 Therefore, certain molecular mechanisms of p16 repression have been directly associated with carcinogenesis. Nevertheless, p16 overexpression has also been reported in a large number of tumors. This overexpression in some types of tumors, such as cervical cancer, perianal lesions, and head and neck cancers, has been associated
FIGURE 1. Case 2: A, Sheets of cohesive epithelioid undifferentiated cells (hematoxylin-eosin; 200). B, Strong and diffused nuclear expression of NUT protein in tumor cells (hematoxylin counterstaining; 400). C, p16 is positive in most of the tumor cells in the cytoplasm and nucleus (hematoxylin counterstaining; 400).
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with infection by high-risk HPV genotypes.3 The presence of HPV oncoproteins E6 and E7 is the molecular mechanism associated with HPV overexpression owing to direct or indirect inactivation of Rb, although there are no known associations with exposures to environmental toxins or infectious agents, smoking, or oncogenic viruses, such as HPV or Epstein-Barr virus, and the development of NC.11 However, p16 overexpression has also been described in the progression of non–HPV-related cancers, such as colon carcinoma and squamous cell carcinoma of the bladder. Those examples have been associated with alterations of the p16-Rb pathway because of mutations silencing the Rb gene of the normal tissue and preneoplastic lesions.3,17 p16 should inhibit proliferation in the cells with a proper functioning p16-Rb pathway. The deregulation of Rb results in the increased p16 expression in the tumor cells because of positive feedback. Rb loss is a frequent event in many neoplasms and it is associated with uncontrolled cell proliferation. Moreover, studies by Romagosa et al3 have provided evidence of the p16 overexpression in a subgroup of undifferentiated highgrade pleomorphic sarcomas, and Rb loss of heterozygosity was found in most of the cases. The negative PCR for HPV in this study indicates that HPV infection does not play a role in the pathogenesis of NC; however, the relationship between p16 and Rb reported in the current literature could explain the overexpression of the p16 tumor-suppressor protein in malignant tumors with uncontrolled proliferation, such as NC. As p16 is one of the most important tumor-suppressor proteins (together with p53), it is a promising target for new studies of anticancer therapies. More research is needed to develop new therapies involving senescence induction by the restoration of p16 functionality. In-depth knowledge of the p16-Rb pathway will be necessary to determine the sensitivity of these therapeutic approaches.3 Moreover, p16 overexpression has been suggested to have a major impact on treatment response and survival in patients with head and neck cancer treated with conventional radiotherapy,3 leading to the hypothesis that malignant tumors overexpressing p16 have increased radiosensitivity. In this sense, radiotherapy could also be an alternative treatment for NC. Nevertheless, no consensus concerning the treatment of NC has been reached because only a limited number of cases have been reported.1 Studies have also suggested epigenomic reprogramming as a potential treatment for NC. Schwartz and colleagues conducted a study in which bulk chromatin acetylation was restored by treating NC cells with histone deacetylase inhibitors, engaging a program of squamous differentiation and arrested growth in vitro that closely mimicked the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of histone deacetylase inhibitor differentiation therapy (the recently FDA-approved vorinor
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stat) was established in 3 different NC xenograft models, where it demonstrated significant growth inhibition and a survival benefit.18 ACKNOWLEDGMENTS The authors thank their colleagues who referred the cases: Daniela Derossi, MD; Carlos Ota´vio Gonc¸alves, MD; and Monica Blaya de Azevedo, MD. The authors also thank Elida B. Ojopi, Lucimara C. Benine, Luciana H. Silva, and Juliana R. Machado, who carried out the PCR evaluation for HPV. REFERENCES 1. Hsieh M, French CA, Liang C, et al. NUT midline carcinoma: case report and review of the literature. Int J Surg Pathol. 2011;19: 808–812. 2. Stelow EB. A review of NUT midline carcinoma. Head and Neck Pathol. 2011;5:31–35. 3. Romagosa C, Simonetti S, Lo´pez-Vicente L, et al. p16Ink4a overexpression overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors. Oncogene. 2011;30:2087–2097. 4. van Dongen JJ, Langerak AW, Bru¨ggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17:2257–2317. 5. de RodaHusman AM, Walboomers JM, van den Brule AJ, et al. The use of general primers GP5 and GP6 elongated at their 30 ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol. 1995;76:1057–1062. 6. Soler C, Allibert P, Chardonnet Y, et al. Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction. J Virol Methods. 1991;35:143–157. 7. van den Brule AJ, Meijer CJ, Bakels V, et al. Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction. J Clin Microbiol. 1990;28:2739–2743. 8. Haack H, Johnson LA, Fry CJ, et al. Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody. Am J Surg Pathol. 2009;33:984–991. 9. French CA, Kutok JL, Faquin WC. Midline carcinoma of children and young adults with NUT rearrangement. J Clin Oncol. 2004;22:4135–4139. 10. Bauer DE, Mitchell CM, Strait KM, et al. Clinicopathologic features and long-term outcomes of NUT midline carcinoma. Clin Cancer Res. 2012;18:5773–5779. 11. French CA. Demystified molecular pathology of NUT midline carcinomas. J Clin Pathol. 2010;63:492–496. 12. French CA, Ramirez CL, Kolmakova J, et al. BRD–NUT oncoproteins: a family of closely related nuclear proteins that M block epithelial differentiation and maintain the growth of carcinoma cells. Oncogene. 2008;27:2237–2242. 13. French CA. Pathogenesis of NUT midline carcinoma. Annu Rev Pathol Mech Dis. 2012;7:247–265. 14. Bishop JA, Westra WH. NUT midline carcinomas of the sinonasal tract. Am J Surg Pathol. 2012;36:1216–1221. 15. Evans AG, French CA, Cameron MJ, et al. Pathologic characteristics of NUT midline carcinoma arising in the mediastinum. Am J Surg Pathol. 2012;36:1222–1227. 16. Gonzalez S, Serrano M. A new mechanism of inactivation of the INK4/ARF locus. Cell Cycle. 2006;5:1382–1384. 17. Alexander RE, Hu Y, Kum JB, et al. p16 expression is not associated with human papillomavirus in urinary bladder squamous cell carcinoma. Mod Pathol. 2012;25:1526–1533. 18. Schwartz BE, Hofer MD, Me Lemieux, et al. Differentiation of NUT midline carcinoma by epigenomic reprogramming. Cancer Res. 2011;71:2686–2696.
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Expression of P16 in NUT Carcinomas With No Association With Human Papillomavirus (HPV) Paulo Guilherme de Oliveira Salles, MD, PhD,* Rafael de Deus Moura, MD,w Letı´cia M. Menezes, MD,* and Carlos E. Bacchi, MD, PhDw
Abstract: NUT carcinoma (NC) is a rare malignant neoplasm usually located in the midline, including the upper aerodigestive tract. NC is an aggressive and highly lethal type of carcinoma. It is defined by the rearrangement of the nuclear protein in the testis (NUT) gene on chromosome 15q14. In most cases, the NUT is involved in a balanced translocation with the BRD4 gene on chromosome 19p13.1, an event that creates a BRD4-NUT fusion gene. The relationship between the human papillomavirus (HPV), p16, and upper aerodigestive tract cancer has been long postulated. In this study, we evaluated the relationship of the p16 expression in 4 cases of NCs and its eventual association with HPV. All 4 cases presented typical histopathologic findings with nuclear positivity of the NUT protein and strong expression for p16. None of these cases, however, showed an association with HPV evaluated by polymerase chain reaction. Despite the expression of p16, this negative result for HPV indicates that HPV infection probably does not play a role in the pathogenesis of NC. Key Words: NUT carcinoma, p16, HPV, immunohistochemistry (Appl Immunohistochem Mol Morphol 2014;22:262–265)
N
UT carcinoma (NC) is a rare, aggressive, and highly lethal cancer that occurs in children and adults of all ages. Most NCs are present in the midline of the upper aerodigestive tract or in the mediastinum as poorly differentiated carcinomas with variable degrees of squamous differentiation. The lung is sometimes involved when tumors are large in size, possibly because of secondary extension; there are only a few reports of primary tumors arising in the lung. Some unusual cases have presented below the diaphragm, including tumors that presented in the posterior bladder and the bones of the pelvis. No cases outside the midline axis have been reported. NC is defined by the rearrangement of the nuclear protein in the testis (NUT) gene on chromosome 15q14. In most cases, NUT is involved in a balanced translocation with the BRD4 gene on chromosome 19p13.1, an event that creReceived for publication May 11, 2013; accepted July 12, 2013. From the *Instituto Mario Penna and Biocor Instituto, Belo Horizonte, MG; and wConsultoria em Patologia, Botucatu, SP, Brazil. The authors declare no conflict of interest. Reprints: Carlos E. Bacchi, MD, PhD, Consultoria em Patologia, Rua Major Leoˆnidas Cardoso 739, Botucatu, SP 18602-010, Brazil (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
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ates a BRD4-NUT fusion gene. Variant rearrangements, some involving the BRD3 gene, occur in the remaining cases. The effect of the NUT protein is not clear; however, BRD4 has a role in transcriptional activation and segregation of the human papillomavirus (HPV) during mitosis. Owing to its rarity and lack of characteristic histologic features, most cases of NC currently go unrecognized, and immunohistochemical analysis using the NUT antibody followed by FISH with probes for the 15q14 NUT break-point may be extremely helpful in the identification of these tumors.1,2 The cyclin-dependent kinase inhibitor 2A (CDKN2A, p16Ink4A), also known as multiple tumor suppressor 1, is a tumor-suppressor protein that plays an important role in regulating the cell cycle, and mutations in p16 increase the risk for developing a variety of cancers. It decelerates the progression of the cell cycle by inactivating the cyclin-dependent kinase that phosphorylates the retinoblastoma protein (pRB). The progression of cells from the G1 phase to the S phase is blocked by protein p16, which acts to disrupt the cyclin D1-CDK4/6 complex. The most critical point in cell-cycle regulation is the G1 checkpoint and it is at this checkpoint that the complex cyclin D1 interactions occur. They determine whether the cell cycle goes back into a quiescent state (G0) or enters the S phase, where cells are destined to divide. When cells enter the S phase, they divide uncontrollably, which leads to cancer.3 A relationship between HPV, p16, and upper aerodigestive tract cancer has long been postulated. HPV-associated squamous cell carcinomas represent a distinct disease entity from carcinogen-associated squamous cell carcinomas. HPV oncoproteins lead to mucosal cell transformation through well-defined mechanisms. The HPV E6 and E7 oncoproteins inactivate the p53 and pRB tumor suppressors, respectively, leading to hyperproliferation and genomic instability. RB inhibits the progression of cells into the S phase and is regulated by cyclin D1 by phosphorylation. Progressive and prolonged phosphorylation of the pRB leads to its inactivation and reduction of its growth suppression activity. This inactivation is mediated by the release of E2F-like transcription factors from RB, which allows the activation of CDK and transcriptional activation of target promoters. The CDKN2A gene product, the p16INK4A protein, inhibits CDK4 and CDK6, which phosphorylate the pRB. A reciprocal relationship between p16INK4A and RB expression
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has been observed. Although the virus is detected in a number of upper aerodigestive tract subsites, studies demonstrate improved outcomes in HPV-associated carcinoma of the oropharynx only. The cell-cycle regulatory protein p16 is upregulated by biologically active HPV and serves as a biomarker of improved response to therapy.3 Our goal was to study the relationship that appears as not yet been described between p16 expression and NCs and the eventual association between this unusual type of carcinoma and HPV.
MATERIALS AND METHODS Case Selection and Pathologic Diagnosis The present series includes 4 cases of NUT midline carcinoma. All cases were retrieved from the archives of Consultoria em Patologia, a large anatomic pathology reference laboratory located in Botucatu, Sa˜o Paulo, Brazil, during the period of April 2010 to January 2012.
Immunohistochemistry Tissues from all cases were formalin fixed and paraffin embedded. The hematoxylin and eosin–stained slides were reviewed for routine morphology. Immunohistochemical analysis was performed using the following panel of antibodies: cytokeratin (clone AE1/AE3, 1:900; Dako); P63 (clone 4A4, 1:4000; Neomarkers); NUT (clone C5B1, 1:100; Cell Signaling); and p16INK (CINtec, 1:200). Novolink polymer (Novocastra, Newcastle upon Tyne, UK) was used as the detection system, and diaminobenzidine was used as the chromogen.
Polymerase Chain Reaction (PCR) for HPV DNA Extraction and Quality Control The hematoxylin and eosin-stained sections of the specimens underwent microdissection before DNA extraction from the formalin-fixed paraffin-embedded tissues. Selected areas and cervical specimens were submitted directly to tissue protein digestion with 100 mg/mL proteinase K for 12 to 15 hours at 551C. DNA was extracted using the saline method. The nucleic-acid purity was determined by a 260/280 nm optical density measurement using a spectrophotometer. A qualitative size range PCR assay was performed to determine the DNA quality of the formalin fixed paraffin embedded tissue and cytologic cervical samples. The assay consisted of 5 primer sets that amplified products of 100, 200, 300, 400, and up to 600 base pairs, and it has previously been used successfully.4
HPV Detection HPV infection was first evaluated by PCR with consensus primers GP5+ and GP6+, which give an amplicon of 138 to 150 base pairs and allow the detection of a broad spectrum of distinct HPV genotypes.5 In addition to undergoing a generic detection for any HPV subtype, the positive samples were submitted to multiplex PCR for discrimination among infections by the main viral genotypes, either of a low risk (HPV 6 and 11) or high risk for cellular transformation (HPV 16, 18, and r
2013 Lippincott Williams & Wilkins
P16 in NUT Carcinoma
33).6,7 All PCR reactions were accomplished with positive and negative controls. The amplified DNA was electrophoresed on 7% polyacrylamide gel. The positive and negative results for the presence of viral DNA obtained through PCR were in agreement with the cytologic and histologic diagnoses.
RESULTS Table 1 summarizes the clinical, immunohistochemical, and molecular features of the NUT midline carcinoma cases. The 4 cases analyzed included 3 men and 1 woman with a mean median age of 31 years (range, 29 to 56 y). Case 1 presented with a nasopharyngeal and ethmoidal sinus tumor with invasion of the orbit. Case 2 presented with a mediastinal mass. In case 3, the patient had multiple nodules in the thorax, central nervous system, parotid gland, lungs, liver, breasts, and adrenal gland. In case 4, the patient had a left lower-lobe lung tumor with multiple cervical and mediastinal lymphadenopathies.
Histologic and Immunohistologic Findings Histologically, all 4 cases were high-grade carcinomas (Fig. 1A), with case 1 showing more overt squamous differentiation and case 4 exhibiting focal areas of keratinization. Cases 2 and 3 were composed of sheets of cohesive undifferentiated epithelioid cells exhibiting vesicular nuclei and prominent nucleoli. Areas of coagulative necrosis, mitotic figures, and apoptotic bodies were frequent in all cases. Glandular differentiation was absent. All 4 cases were diffusely positive for cytokeratin and NUT (Fig. 1B) and at least focally positive for p63. p16 was expressed in all cases in at least 50% of the tumor cells (Fig. 1C). All 4 cases were negative for HPV by PCR.
DISCUSSION NUT midline carcinoma is a rare type of malignant epithelial tumor defined by its typical rearrangement on chromosome 15q14 of the gene encoding the nuclear protein of the testis (NUT).2 NUT expression is normally confined to the germ cells of the testis and ovary and has not been detected in human tumors other than NC.8 The characteristics of NC were first described in 2004,9 and the largest cohort of patients with NC studied to date included 63 patients. NC typically arises in the midline head and neck structures or in the thorax and shows an aggressive behavior with early regional invasion and distant metastasis.10 Initially, it was thought to be a childhood cancer most likely because of a selection bias, as cytogenetic analysis and karyotypes are more often obtained for children’s tumors. More recent studies have shown that NC can affect people of all ages with no predilection for either sex.11 In contrast to most carcinomas, which commonly have complex chromosomal abnormalities, NC typically has simple karyotypes, and in some cases the only aberration is the chromosomal rearrangement involving www.appliedimmunohist.com |
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TABLE 1. Summary of the Clinical, Immunohistochemical, and Molecular Features of the 4 Cases of NUT Midline Carcinoma Age (y) Sex Anatomic location
Cytokeratin p63 NUT p16 HPV-PCR
Case 1
Case 2
Case 3
Case 4
30 Male Nasopharynx/ ethmoidal sinus
29 Male Mediastinum
56 Male Lung and lymph nodes
+ + + +
+ + + +
34 Female Thorax, central nervous system, parotid gland, lungs, liver, breasts, and adrenal + + + +
+ F + +
F indicates focally positive (< 50%); HPV, human papillomavirus; PCR, polymerase chain reaction.
NUT. Those simple karyotypes are typically more present in leukemias, lymphomas, and sarcomas. Considering the pathogenesis of these tumor types, it has been hypothesized that NUT fusion proteins may initiate malignant transformation within epithelial cell precursors and require relatively few collaborative mutations or epigenetic changes to produce NC.12 Morphologically, NCs are indistinguishable from other poorly differentiated carcinomas. Histologically, they are composed of sheets of undifferentiated cells with scant amphophilic or eosinophilic cytoplasm. Occasional cases may have more nested malignant cells within a desmoplastic stroma. An abrupt squamous differentiation is frequently observed with maturing squamous cells and extracellular keratin formation. The cell nuclei have irregular contours, but they tend to have a uniform size with prominent nucleoli and fine vesicular chromatin.2 NC has recently become diagnosable in most cases by immunohistochemical staining with an anti-NUT monoclonal antibody. Improvements in diagnostic methods have led to increased recognition of NC, but its true prevalence remains unknown.13 Few retrospective studies have been conducted concerning the epidemiology of NC. In a study conducted at The Johns Hopkins Hospital, the archival surgical pathology files were searched for all cases of primary sinonasal carcinomas diagnosed from 1995 to 2011; of the 151 cases, only 3 (2%) were NUT positive.14 Evans and colleagues examined the NUT expression in 114 cases of poorly differentiated carcinomas or unclassified
mediastinal malignancies. Four of their cases (3.5%) showed positivity for the NUT expression. Therefore, NC should be considered in the differential diagnosis of any poorly differentiated epithelioid mediastinal tumor, regardless of age.15 Three of 4 of our cases were immunoreactive to p63 antibodies—consistent with squamous differentiation—and all of them were positive for cytokeratin. Interestingly, all 4 cases were also immunoreactive to p16, and despite the presence of p16 expression, none of these tumors showed evidence of HPV infection measured by PCR. p16 is a protein involved in the negative regulation of cell proliferation and is therefore considered a tumor suppressor.3 According to Gonzalez and Serrano,16 close to 50% of all human cancers show p16 inactivation ranging from 25% to 70%. These include head and neck, esophageal, biliary tract, liver, lung, and breast carcinomas. It is well known that p16 contributes to the regulation of cell-cycle progression by inhibiting the S phase. The expression of p16 maintains the Rb family members in a hypophosphorylated state, which promotes binding to E2F1 and leads to G1 cell-cycle arrest.3 Therefore, certain molecular mechanisms of p16 repression have been directly associated with carcinogenesis. Nevertheless, p16 overexpression has also been reported in a large number of tumors. This overexpression in some types of tumors, such as cervical cancer, perianal lesions, and head and neck cancers, has been associated
FIGURE 1. Case 2: A, Sheets of cohesive epithelioid undifferentiated cells (hematoxylin-eosin; 200). B, Strong and diffused nuclear expression of NUT protein in tumor cells (hematoxylin counterstaining; 400). C, p16 is positive in most of the tumor cells in the cytoplasm and nucleus (hematoxylin counterstaining; 400).
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with infection by high-risk HPV genotypes.3 The presence of HPV oncoproteins E6 and E7 is the molecular mechanism associated with HPV overexpression owing to direct or indirect inactivation of Rb, although there are no known associations with exposures to environmental toxins or infectious agents, smoking, or oncogenic viruses, such as HPV or Epstein-Barr virus, and the development of NC.11 However, p16 overexpression has also been described in the progression of non–HPV-related cancers, such as colon carcinoma and squamous cell carcinoma of the bladder. Those examples have been associated with alterations of the p16-Rb pathway because of mutations silencing the Rb gene of the normal tissue and preneoplastic lesions.3,17 p16 should inhibit proliferation in the cells with a proper functioning p16-Rb pathway. The deregulation of Rb results in the increased p16 expression in the tumor cells because of positive feedback. Rb loss is a frequent event in many neoplasms and it is associated with uncontrolled cell proliferation. Moreover, studies by Romagosa et al3 have provided evidence of the p16 overexpression in a subgroup of undifferentiated highgrade pleomorphic sarcomas, and Rb loss of heterozygosity was found in most of the cases. The negative PCR for HPV in this study indicates that HPV infection does not play a role in the pathogenesis of NC; however, the relationship between p16 and Rb reported in the current literature could explain the overexpression of the p16 tumor-suppressor protein in malignant tumors with uncontrolled proliferation, such as NC. As p16 is one of the most important tumor-suppressor proteins (together with p53), it is a promising target for new studies of anticancer therapies. More research is needed to develop new therapies involving senescence induction by the restoration of p16 functionality. In-depth knowledge of the p16-Rb pathway will be necessary to determine the sensitivity of these therapeutic approaches.3 Moreover, p16 overexpression has been suggested to have a major impact on treatment response and survival in patients with head and neck cancer treated with conventional radiotherapy,3 leading to the hypothesis that malignant tumors overexpressing p16 have increased radiosensitivity. In this sense, radiotherapy could also be an alternative treatment for NC. Nevertheless, no consensus concerning the treatment of NC has been reached because only a limited number of cases have been reported.1 Studies have also suggested epigenomic reprogramming as a potential treatment for NC. Schwartz and colleagues conducted a study in which bulk chromatin acetylation was restored by treating NC cells with histone deacetylase inhibitors, engaging a program of squamous differentiation and arrested growth in vitro that closely mimicked the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of histone deacetylase inhibitor differentiation therapy (the recently FDA-approved vorinor
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stat) was established in 3 different NC xenograft models, where it demonstrated significant growth inhibition and a survival benefit.18 ACKNOWLEDGMENTS The authors thank their colleagues who referred the cases: Daniela Derossi, MD; Carlos Ota´vio Gonc¸alves, MD; and Monica Blaya de Azevedo, MD. The authors also thank Elida B. Ojopi, Lucimara C. Benine, Luciana H. Silva, and Juliana R. Machado, who carried out the PCR evaluation for HPV. REFERENCES 1. Hsieh M, French CA, Liang C, et al. NUT midline carcinoma: case report and review of the literature. Int J Surg Pathol. 2011;19: 808–812. 2. Stelow EB. A review of NUT midline carcinoma. Head and Neck Pathol. 2011;5:31–35. 3. Romagosa C, Simonetti S, Lo´pez-Vicente L, et al. p16Ink4a overexpression overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors. Oncogene. 2011;30:2087–2097. 4. van Dongen JJ, Langerak AW, Bru¨ggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17:2257–2317. 5. de RodaHusman AM, Walboomers JM, van den Brule AJ, et al. The use of general primers GP5 and GP6 elongated at their 30 ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol. 1995;76:1057–1062. 6. Soler C, Allibert P, Chardonnet Y, et al. Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction. J Virol Methods. 1991;35:143–157. 7. van den Brule AJ, Meijer CJ, Bakels V, et al. Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction. J Clin Microbiol. 1990;28:2739–2743. 8. Haack H, Johnson LA, Fry CJ, et al. Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody. Am J Surg Pathol. 2009;33:984–991. 9. French CA, Kutok JL, Faquin WC. Midline carcinoma of children and young adults with NUT rearrangement. J Clin Oncol. 2004;22:4135–4139. 10. Bauer DE, Mitchell CM, Strait KM, et al. Clinicopathologic features and long-term outcomes of NUT midline carcinoma. Clin Cancer Res. 2012;18:5773–5779. 11. French CA. Demystified molecular pathology of NUT midline carcinomas. J Clin Pathol. 2010;63:492–496. 12. French CA, Ramirez CL, Kolmakova J, et al. BRD–NUT oncoproteins: a family of closely related nuclear proteins that M block epithelial differentiation and maintain the growth of carcinoma cells. Oncogene. 2008;27:2237–2242. 13. French CA. Pathogenesis of NUT midline carcinoma. Annu Rev Pathol Mech Dis. 2012;7:247–265. 14. Bishop JA, Westra WH. NUT midline carcinomas of the sinonasal tract. Am J Surg Pathol. 2012;36:1216–1221. 15. Evans AG, French CA, Cameron MJ, et al. Pathologic characteristics of NUT midline carcinoma arising in the mediastinum. Am J Surg Pathol. 2012;36:1222–1227. 16. Gonzalez S, Serrano M. A new mechanism of inactivation of the INK4/ARF locus. Cell Cycle. 2006;5:1382–1384. 17. Alexander RE, Hu Y, Kum JB, et al. p16 expression is not associated with human papillomavirus in urinary bladder squamous cell carcinoma. Mod Pathol. 2012;25:1526–1533. 18. Schwartz BE, Hofer MD, Me Lemieux, et al. Differentiation of NUT midline carcinoma by epigenomic reprogramming. Cancer Res. 2011;71:2686–2696.
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