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Keywords:

  • NUT midline carcinoma;
  • aggressive carcinoma;
  • undifferentiated carcinoma;
  • t(15;19);
  • BRD4;
  • cytology;
  • fine-needle aspiration;
  • immunohistochemistry;
  • fluorescent in situ hybridization;
  • differential diagnosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

BACKGROUND:

Nuclear protein in testis (NUT) midline carcinoma (NMC) represents an aggressive, high-grade carcinoma typically involving the upper aerodigestive tract or mediastinum. Although the tumor was originally noted in young persons, we have subsequently identified 5 adult cases. To our knowledge, the cytology of NMC has not been systematically described.

METHODS:

We recently published a series of NMCs identified by fluorescent in situ hybridization for characteristic NUT rearrangement. Three of these patients had undergone fine-needle aspiration. Patient age, sex, primary tumor location, and aspiration site were noted. Cases were assessed for the following: cellularity, architecture, cytoplasm, cell size, nuclear contours, nucleoli, chromatin, anisonucleosis/cytosis, mitotic activity, background, and nuclear crush.

RESULTS:

The 3 cases occurred in 2 women and 1 man, ages 31-79 years. Primaries involved the sinonasal tract (2) and larynx. Aspirates were of right neck masses (2) and supraclavicular lymph node. Smears were highly cellular and generally noncohesive. Cytoplasm was scant/delicate, although occasional cells with denser cytoplasm were noted in 1 case. Cells were 2-3 times the diameter of a small lymphocyte with irregular nuclear contours, discrete nucleoli, and fine/granular to vesicular chromatin. Anisonucleosis/cytosis was slight to moderate. Mitotic figures were noted in each case. The background contained naked nuclei and karyorrhectic debris; nuclear crush was noted.

CONCLUSIONS:

NMCs exhibit cytologic features of a poorly differentiated or undifferentiated carcinoma. Although reports mention squamous differentiation as a histologic feature, it is typically focal, and overt squamous differentiation was not identified in our cases. Given morphologic overlap with other high-grade carcinomas, diagnosis requires a high index of suspicion. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.

Nuclear protein in testis (NUT) midline carcinoma (NMC) represents a rare, clinically aggressive, and high-grade carcinoma molecularly defined by translocation involving the NUT gene on chromosome 15. In 1991, two groups independently published case reports of intrathoracic high-grade carcinomas in young persons with a novel translocation t(15;19).1, 2 Several reports of single cases and 2 small series followed.3-10 Lesions involved young patients (median age 16 years), typically presented in the head and neck or thorax, were undifferentiated or poorly differentiated with focal squamous differentiation and were associated with survivals measured in months.

The tumor has been reported under various names including “midline carcinoma of children and young adults” and “aggressive carcinoma with translocation t(15;19)” and is included in the 2004 World Health Organization tumor blue book of the lung, pleura, thymus, and heart under the heading “carcinoma with t(15;19) translocation.”5, 8, 11 The characteristic translocation results in fusion of the 5′ aspect of BRD4 on chromosome 19 with nearly the entire coding region of NUT.5, 12 The development of a fluorescent in situ hybridization (FISH) assay for the NUT translocation led to the discovery of alternative fusion partners (including BRD3 on chromosome 9).8, 13 The lesion's current designation, NUT midline carcinoma, reflects this finding.

We recently reported a series of 5 NMCs identified by FISH screening a group of undifferentiated carcinomas from the upper aerodigestive tract.14 Although previous screening attempts had been undertaken in pediatric tumors, our patient population was unselected for age. All 5 tumors involved mature adults, ages 31-78 years, including 4 patients older than any previously reported. Three of these patients had undergone fine-needle aspiration.

The aim of this study is to describe the cytologic features of NMC with an emphasis on differential diagnosis. We conclude that given considerable morphologic overlap with other poorly differentiated tumors and the prior notion that it represented a “pediatric tumor,” most cases presently go undiagnosed. Awareness of this entity, including its characteristic although not diagnostic clinicopathologic features, would allow for the ancillary testing necessary to secure the diagnosis. To our knowledge this represents the first report of NUT midline carcinoma in the cytology literature.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

We recently reported a series of 5 NMCs identified by testing a cohort of 28 EBV-negative, high-grade carcinomas from the sinonasal tract, nasopharynx, and larynx for disruption of BRD4 and/or NUT. Original histologic diagnoses from the larger cohort included sinonasal undifferentiated carcinoma (SNUC), undifferentiated carcinoma (not otherwise specified), and poorly differentiated carcinoma with focal squamous differentiation.14 In 3 of these 5 cases, cytologic material (ethanol-fixed, Papanicolaou-stained smears derived from fine-needle aspiration of metastases to superficial sites) was available for present review. NUT, p63 immunohistochemistry, and periodic acid-Schiff (PAS) with and without diastase and mucicarmine histochemistry had been performed previously on corresponding surgical pathology material.14, 15 Patient age, sex, primary tumor site, aspirated site, and original cytologic diagnosis were noted; concise clinical follow-up was obtained. Cases were assessed for the following cytologic features: cellularity, architecture, cytoplasm, cell size, nuclear contours, nucleoli, chromatin, anisonucleosis/cytosis, mitotic activity, background, and nuclear crush.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

The 3 cases occurred in 2 women and 1 man, ages 31-79 years (Figs. 1-3). Two primaries involved the sinonasal tract and 1 involved the larynx. Aspirates were of 2 right neck masses and 1 supraclavicular lymph node. Original cytologic diagnoses included “poorly differentiated carcinoma consistent with SNUC,” “metastatic SNUC,” and “poorly differentiated malignant neoplasm consistent with carcinoma.” Patients were treated with combinations of chemoradiation and surgery. All patients died of disease in less than 1 year. These findings are summarized in Table 1.

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Figure 1. Case 1. (A) Surgical biopsy of the nasal cavity lesion demonstrates nests of epithelioid cells with prominent central necrosis. Squamous differentiation is absent (H & E ×200). (B) Aspiration of the right neck mass reveals a loosely cohesive epithelial sheet; small nucleoli are identified (Papanicolaou ×400). (C) Rare punched out spaces (arrow) are negative on mucin stains; karyorrhectic material (circle) is noted (Papanicolaou ×600).

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Figure 2. Case 2. (A) Surgical biopsy of the nasal cavity/frontal sinus mass reveals nests of undifferentiated epithelioid cells with abrupt keratinization; this is the classically described histologic finding in nuclear protein in testis midline carcinoma (H & E ×400). (Inset: This lesion exhibits diffuse, strong p63 nuclear immunoreactivity; immunoperoxidase, ×100). (B) Although aspiration of the supraclavicular lymph node lesion fails to reveal overt squamous differentiation, occasional cells exhibit dense cytoplasm (Papanicolaou ×600). (C) The aspirate demonstrates a noncohesive smear pattern with anisonucleosis of generally 1.5-2× but with occasional larger cells (arrow) noted (Papanicolaou ×600).

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Figure 3. Case 3. (A) Resection of this supraglottic laryngeal mass displays sheets of noncohesive intermediate-sized cells with vesicular chromatin, prominent nucleoli, and frequent mitotic figures; squamous differentiation is again absent (H & E ×400). (B) Aspiration of the right neck mass is highly cellular with isolated cells greatly predominating, cytoplasm is scant and very delicate, and the smear pattern evokes the differential diagnosis of lymphoma (Papanicolaou ×600).

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Table 1. Clinical Features of NMC Cases
 AgeSexPrimary SiteAspirated SiteOriginal Cytologic DiagnosisTreatmentFollow-Up
  1. NMC indicates nuclear protein in testis midline carcinoma; R, right; PDC, poorly differentiated carcinoma; C/W, consistent with; SNUC, sinonasal undifferentiated carcinoma; DOD, died of disease; LN, lymph node; CA, carcinoma.

Case 131MNasal cavityR neckPDC c/w SNUC• ChemoradiationDOD at 10 mo
• Craniofacial resection
• Neck dissection
Case 239FNasal cavity and frontal sinusSupraclavicular LNMetastatic SNUC• ChemoradiationDOD at 7 mo
• Craniofacial resection
Case 378FSupraglottic larynxR neckPoorly differentiated malignant neoplasm c/w metastatic CA• ChemoradiationDOD at 8 mo
• Laryngectomy
• Neck dissection

Smears were highly cellular and noncohesive. In Case 1, although single cells and small, loose clusters predominated, occasional large sheets were noted (Fig. 1b). Case 3 was comprised nearly entirely of single cells (Fig. 3b). Rare extracellular “lumina” were identified in Case 1 and occasional cytoplasmic vacuoles were seen in cases 1 and 2 (Fig. 1c). Cytoplasm was scant and delicate, although occasional cells with denser cytoplasm were noted in Case 2 (Fig. 2b). Cells were 2-3 times the diameter of a small lymphocyte with occasional larger cells noted (Fig. 2c). Nuclear contours were somewhat irregular and nucleoli were readily identifiable and variably prominent. Chromatin ranged from fine/granular to vesicular; in no case was it overtly neuroendocrine. Anisonucleosis/cytosis was slight to moderate, with variation in nuclear area in any individual case ranging from 1.5 to 3. Mitotic figures were noted in each case and were numerous in Case 3. The background contained naked nuclei and karyorrhectic debris; nuclear crush was noted. The cytologic features of NMC are summarized in Table 2.

Table 2. Cytologic Features of NMC
  1. NMC indicates nuclear protein in testis midline carcinoma.

• Highly cellular smears
• Noncohesive with prominent single cells
• Scant, delicate cytoplasm; occasional cells with dense cytoplasm possible
• Rare cytoplasmic vacuoles and extracytoplasmic lumina (negative for epithelial mucin)
• Cells 2-3 times the diameter of a small lymphocyte
• Irregular nuclear contours
• Identifiable, variably prominent nucleoli
• Fine/granular or vesicular chromatin
• Slight to moderate anisocytosis and anisonucleosis
• Identifiable mitotic figures (may be prominent)
• Background with naked nuclei and karyorrhectic debris
• Possible nuclear crush

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

NUT midline carcinoma demonstrates the cytologic features of a poorly differentiated to undifferentiated malignant neoplasm. In 2 of our cases, the presence of at least focal cellular cohesion supported the interpretation of carcinoma. In the final case, that determination required correlation with the patient's known primary lesion.

Foci of abrupt keratinization in an otherwise undifferentiated carcinoma are typical of NMC; they are especially prominent in so-called NUT-variant cases (in which the NUT translocation partner is not BRD4) (Fig. 2a).8, 13 That overt keratinization was not observed in any of our cases may reflect bias in our case selection, although in our experience, the keratinization in surgical material is often very focal. The occasional cells with dense cytoplasm in Case 2 hint at squamous differentiation. And, in theory, overt keratinization may be identifiable. The extracellular lumina and cytoplasmic vacuoles observed in cases 1 and 2 have previously been shown to reflect a combination of degenerative changes and glycogen.15 In fact, in the 21 NMCs reported to date, focal glandular differentiation has been reported only in 1.3

Findings in cell block material that would support a diagnosis of NMC include immunoreactivity with antibodies directed against pankeratin, p63, and CD34. We recently reported p63-positivity in 5 of 5 cases and speculate that this reflects the underlying squamous nature of this tumor.14 Surprising for an epithelial tumor, in one series CD34 immunoreactivity was reported in 7 of 11 NMCs. This may reflect the primitive or undifferentiated nature of many of the neoplastic cells, and no significant staining was observed in a control group of 45 carcinomas in patients < 40 years old, including 11 lymphoepitheliomas.8 Pertinent negatives include lack of reactivity for Epstein-Barr virus (EBV), CD45, S-100, and neuroendocrine markers. Focal, weak placental alkaline phosphatase (PLAP) reactivity has been noted in 1 case.8 The anticipated immunophenotypes of NMC and relevant differential diagnostic entities are summarized in Table 3. It is worth noting p63's lack of specificity in the diagnostic workup of a poorly differentiated carcinoma of the head and neck, as it is typically also expressed in nasopharyngeal carcinoma and occasionally in SNUC and small-cell carcinoma.

Table 3. Selected Immunohistochemistry of NMC and Differential Diagnostic Entities
 CKp63NUTEBV*S-100LCAPLAPCD56
  • NMC indicates nuclear protein in testis midline carcinoma; NUT, nuclear protein in testis; SNUC, sinonasal undifferentiated carcinoma; NPC, nasopharyngeal carcinoma; SCNC, small-cell neuroendocrine (undifferentiated) carcinoma; GCT, germ-cell tumor; MM, malignant melanoma; CK, (pan)cytokeratin; EBV, Epstein-Barr virus; LCA, leukocyte common antigen; PLAP, placental alkaline phosphatase; +, positive; +/-, often positive; -/+, occasionally positive; R+, rarely reported positive; -, negative; U, unknown.

  • *

    In-situ hybridization for Epstein-Barr virus-encoded RNA increases sensitivity over immunohistochemical detection of latent membrane protein .

  • Dependent on the histologic subtype.

NMC+++R+
SNUC+−/+−/+
NPC+++R+
SCNC+/−−/+R+R++/−
GCT−/+−/+−/++/−
Hematolymphoid neoplasmR+U+
MMR+R+U+R+

NMC should be considered in the differential diagnosis of a variety of poorly differentiated and undifferentiated carcinomas. Given our experience, the distinction of NMC from SNUC represents a challenging differential, as they exhibit wholly overlapping cytologic features.15 Ejaz et al recently advocated including largely undifferentiated sinonasal tumors with rare foci of squamous differentiation under the diagnostic umbrella of SNUC; we would expect that a proportion of such cases would have demonstrable NUT translocations.16 Immunohistochemical demonstration of a neuroendocrine phenotype (identified in a minority of SNUCs) may effectively exclude NMC.14 SNUC and NMC exhibit similarly dismal prognoses.17

Undifferentiated nasopharyngeal carcinoma (NPC) represents the “can't miss” diagnosis in this differential. A significant proportion of these lesions exhibit a prominent lymphoid stroma (so-called “lymphoepithelioma”), and aspirates demonstrate a dual population of high-grade carcinoma cells and lymphoid material. Other associated findings may include the presence of epithelioid granulomas and prominent eosinophils.18 Although none of our cases exhibited this dual population, Vargas and colleagues reported a surgical case in which a prominent lymphoid infiltrate simulated lymphoepithelioma.6 Typical clinical features include a predilection for Southeast Asians and a tendency to present with cervical lymph node metastases in the face of an occult primary. Demonstration of EBV infection is critical to diagnosis. The lesion demonstrates a distinctly better prognosis and is often treated with radiation alone.19

The anatomic distribution of NMC and small-cell neuroendocrine carcinoma (SCNC) overlap. SCNC may present in the anterior mediastinum either as a metastasis from a lung primary or as a thymic primary, and rare sinonasal primary tumors have been described.20, 21 Compared with SCNC, an NMC would be expected to demonstrate larger cell size, more voluminous cytoplasm, and discrete nucleoli, and it would lack overtly neuroendocrine chromatin. Furthermore, we failed to observe the degree of nuclear molding typical of SCNC. Demonstration of a neuroendocrine phenotype by immunohistochemistry is a reasonable expectation in SCNC (although not a universal finding) and would likely exclude NMC. Although SCNC shares a poor prognosis, this distinction is important as patients may derive at least initial benefit from “small-cell chemotherapy” (i.e. combination of etoposide and platinum-based agent).22

The isolated cell pattern observed in our Case 3 evokes a broader differential diagnosis beyond that of other poorly to undifferentiated carcinomas. Seminoma may present in the anterior mediastinum and would produce a similar smear pattern. A dual cell population (large epithelioid cells admixed with small lymphocytes, similar to that seen in lymphoepithelioma) is characteristic, and although not encountered in our few cases, as described above, it has been encountered in surgical material. The classic “tigroid” background (attributable to smearing of fragile cytoplasmic elements) is usual, particularly in air-dried material. Given that we had access only to ethanol-fixed material in this study, we cannot exclude the possibility that some NMCs could demonstrate a similar background. In suspect cases, we use PLAP as an immunohistochemical screen.

A hematolymphoid neoplasm would also produce a cellular, noncohesive smear. And, in fact, the cell size, chromatin characteristics, and prominent nuclei in our NMCs are particularly reminiscent of large cell lymphoma. Lymphoglandular bodies, not identified in our NMCs, are potentially useful in the discrimination of a noncohesive, high-grade carcinoma from a lymphoma. Most hematolymphoid neoplasms will express CD45. More extensive immunophenotyping is directed by the specific clinical and morphologic features of the case.

Finally, melanoma would represent a consideration. Cytologic features of melanoma not observed in our NMCs include a mixed epithelioid and spindle cell pattern, prominent intranuclear pseudoinclusions, and, of course, melanin pigment. Most melanomas express S-100 protein, while NMCs do not.

Ultimately, the diagnosis of NMC rests on the demonstration of the diagnostic NUT translocation. This can be accomplished by various methods including conventional karyotyping, reverse transcription polymerase chain reaction (RT-PCR), and FISH. All of these are possible on fresh aspirate material with the final 2 still possible in formalin-fixed, paraffin-embedded cell block material. FISH and conventional karyotyping have the advantage of being able to detect NUT-variant cases. Unfortunately, these specific RT-PCR and FISH assays are not available from clinical laboratories, performed instead by a few research laboratories.23

The development of antibodies to NUT for use in routine immunohistochemistry holds promise to bring the diagnosis of NMC to the diagnostic surgical pathology laboratory, allowing for selection of cases for confirmatory FISH testing. A monoclonal antibody in development achieved a sensitivity of 87% and a specificity of 100% in a large group of nongerm cell tumors.24 NUT immunohistochemistry takes advantage of the finding that the normal NUT protein demonstrates extremely restricted expression (limited to the testis and ciliary ganglion).12, 23 Ectopic expression in NMC appears attributable to NUT's placement under control of the promoter elements of the ubiquitously expressed BRD4 (and other related genes in NUT-variant cases).

Although, at present, the distinction of NMC from other EBV-negative poorly differentiated to undifferentiated carcinomas is mainly academic, the development of an effective directed biologic therapy would make the distinction essential. NMCs are genetically simple, with NUT translocation generally the lone demonstrable karyotypic abnormality.1-3, 6, 7, 9, 10 Given this, it can be assumed that the resulting fusion protein is pathogenetically fundamental. This hypothesis is given credence by recent siRNA experiments in NMC cell lines, in which BRD4-NUT knockdown resulted in striking squamous differentiation and growth arrest.13 An effective therapeutic could either reduce fusion protein levels or interfere with the protein's function. BRD4 has been shown to associate with acetylated chromatin and likely serves the same function in the BRD4-NUT fusion.25 Disruption of this interaction would possibly obviate the effects of the fusion protein.

Conclusions

NMC demonstrates the cytologic features of a poorly differentiated to undifferentiated carcinoma. Squamous differentiation, typical in surgical pathology specimens, may not be observed. NMC should be considered in the differential diagnosis of any EBV-negative poorly differentiated to undifferentiated carcinoma that lacks glandular or neuroendocrine differentiation. A panel of immunohistochemical stains may assist in the differential diagnosis. Definitive diagnosis requires demonstration of NUT translocation, which may be accomplished by conventional karyotyping, RT-PCR, or FISH. NUT immunohistochemistry is under development and could serve as an initial screening tool. At present, the diagnosis of NMC, other than connoting a particularly poor prognosis, is mainly academic. Given the development of a directed biologic therapy, targeting the fusion protein, the distinction would become essential.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References