1 Introduction
Nuclear protein of the testis (NUT) midline carcinoma (NMC) is a rare and molecularly defined malignancy that represents a distinct clinicopathologic entity. Its defining molecular hallmark is chromosomal rearrangements involving the NUTM1 gene, located at 15q14 [
1]. This aggressive epithelial neoplasm typically exhibits histologic features of poorly differentiated squamous carcinoma and demonstrates a predilection for midline structures, including the head, neck, thorax, and mediastinum. However, emerging evidence has documented extra-midline presentations, affecting diverse anatomical sites such as the pulmonary parenchyma, viscera, and soft tissues [
2,
3]. Epidemiologic analyses reveal a median age of onset between 16 and 24 years, although cases have been reported across pediatric to geriatric populations, highlighting its pan-age pathogenic potential [
4].
Primary pulmonary NMC, recognized in the WHO classification of thoracic tumors [
5], is associated with an exceptionally poor prognosis. The median overall survival (OS) is 6.7 months (interquartile range: 6–9 months), and the 2-year survival rate is below 20% [
6]. While surgical resection can improve outcomes in early-stage disease, with progression-free survival (PFS) exceeding 12 months [
7], most patients present with advanced metastatic disease at diagnosis, involving lymphatic, osseous, and pleural sites. This advanced presentation often precludes curative intent surgery [
8]. Conventional multimodal therapy, combining platinum-based chemotherapy and radiotherapy, has shown limited clinical benefit, with rapid therapeutic resistance observed in most cases.
Recent insights into tumor vasculature dynamics have proposed anti-angiogenic strategies as potential therapeutic adjuvants for NMC. Mechanistically, inhibiting angiogenesis may normalize tumor vasculature, enhancing intratumoral drug delivery and potentiating synergistic effects with cytotoxic agents, immunotherapies, or molecularly targeted therapies [
9]. This paradigm has been well-established in the management of non-small cell lung cancer (NSCLC) [
10,
11], but it remains underexplored in NMC. To date, only anecdotal reports have documented the application of anti-angiogenic therapy in this context.
In this study, we present two cases of advanced primary pulmonary NMC managed with a combination of anti-angiogenic agents, conventional chemotherapy, and radiotherapy. Our cohort demonstrated favorable survival outcomes, with PFS of 15 and 7 months, and OS of 32 and 13 months, respectively. These results not only challenge the therapeutic nihilism surrounding this malignancy but also provide a compelling rationale for incorporating anti-angiogenic targeted therapy into the management of NMC.
2 Case presentation
2.1 Case 1
A 31-year-old male with no smoking history presented in April 2021 with a 1-month history of persistent cough. Thoracoabdominal contrast-enhanced CT identified a 4.7 cm × 3.4 cm heterogeneously enhancing mass at the right middle lobe bronchial root, accompanied by multistation mediastinal (2R, 4R/L, 7) and hilar lymphadenopathy (short-axis diameter 1.6 cm). Distal pulmonary lesions (maximum 2.8 cm × 2.5 cm) and left gastric lymph node enlargement raised suspicion for metastatic dissemination (Fig.1). Initial staging via brain MRI and bone scintigraphy showed no distant involvement.
Transbronchial biopsy revealed sheets of poorly differentiated epithelioid cells with focal squamous differentiation (typical keratin pearls) and high-grade cytomorphology: hyperchromatic nuclei, prominent nucleoli, and brisk mitotic activity (Fig.2 and 2B). Immunohistochemical profiling demonstrated diffuse nuclear NUT expression, cytokeratin co-expression (CK5/6, CK7, p40), and negative neuroendocrine/thyroid markers (TTF-1, Syn, CgA, CD56). Tumor mutational burden was low (4.8 mutations/Mb), with PD-L1 negativity (TPS 0%) and Ki-67 proliferation index of 40% (Fig.2 and 2D). Next-generation sequencing (NGS) confirmed BRD4-NUTM1 rearrangement, securing the diagnosis of stage IVA (cT3N3M1b) primary pulmonary NMC.
First-line therapy with albumin-bound paclitaxel plus cisplatin administered over six cycles (May–October 2021) achieved partial response (PR) by RECIST 1.1 criteria. Concurrent image-guided radiotherapy (50 Gy/25 fractions) to the left gastric lymph nodes achieved locoregional control (Fig.1). Despite initial response, anti-angiogenic therapy with anlotinib was initiated in December 2021 given the tumor’s aggressive biology. Serial imaging demonstrated stable disease (SD) until August 2022, when hepatic metastasis (T1bN3M1c, IVB) and primary tumor progression necessitated therapy discontinuation, yielding a first-line PFS of 15 months (Fig.3).
The patient entered a phase I trial of the BET inhibitor (BETi) NHWD-870 in January 2023. Rapid skeletal progression (L3 vertebral destruction) developed at 4 months, prompting palliative spinal radiotherapy (30 Gy/10 fractions). Subsequent third-line therapy with sintilimab, etoposide, and carboplatin failed to halt disease advancement. Fourth-line histone deacetylase inhibitor (HDAC-I) tucidinostat combined with doxorubicin similarly showed minimal efficacy in December 2023. The patient ultimately succumbed to multiple organ dysfunction syndrome (MODS) in January 2024, achieving an OS of 32 months—4.8-fold longer than the reported median OS for advanced NMC.
2.2 Case 2
A 26-year-old non-smoking female presented with incidental imaging findings of non-tender lower abdominal masses during routine health screening. Referral CT scans demonstrated a 10.0 cm × 5.5 cm lobulated mass in the right middle-lower lung lobe with bronchial root involvement, ipsilateral hilar/mediastinal lymphadenopathy (short-axis 2.8 cm), and bilateral adnexal/pelvic metastases (dominant lesion 22 cm × 13 cm) (Fig.1). Baseline metastatic brain MRI and bone scintigraphy revealed no further dissemination.
Histopathological evaluation of transbronchial and pelvic biopsies revealed poorly differentiated carcinoma with geographic necrosis, neutrophilic infiltrates, and focal keratinization (Fig.4 and 4B). Immunohistochemistry confirmed nuclear NUT expression, squamous differentiation markers (CK5/6, p40, p63), and retained INI1 expression, while excluding neuroendocrine/thyroid lineage (Syn, CgA, TTF-1 negative) (Fig.4 and 4D). FISH confirmed NUTM1 rearrangement, establishing the diagnosis of stage IVB (cT4N2M1c) primary pulmonary NMC with gynecological metastasis.
After pathological consultation in our hospital, the patient was treated with tislelizumab, paclitaxel liposome, and nedaplatin for one cycle. After one cycle of treatment, the patient experienced significant weight loss (5 kg in 2 weeks), accompanied by fatigue and muscle aches. Subsequent CT scans showed that the mass in the right middle lower lobe bronchus, measuring up to 10 cm × 5.5 cm, had merged with the enlarged lymph nodes in the right hilum and mediastinum. Additionally, there were multiple masses in the bilateral uterine adnexa, abdomen, and pelvic cavity, with the largest measuring 22 cm × 13.0 cm. After anti-inflammatory treatment, the lesion did not decrease in size, and the patient subsequently changed the treatment regimen.
As the second-line treatment, the patient received a total of 6 cycles of chemotherapy and anti-angiogenic therapy with paclitaxel-albumin conjugate, cisplatin, and bevacizumab. PR was achieved after the first 2 cycles of treatment (Fig.1D). The patient’s lesions remained stable after 6 cycles of treatment. Subsequently, the patient received 2 cycles of maintenance therapy with paclitaxel-albumin conjugate and bevacizumab. However, CT scan revealed newly developed multiple enlarged lymph nodes in the right hilum (short diameter: approximately 2.8 cm), and the PFS was 7 months.
Hypofractionated radiotherapy (37.5 Gy/15 fx) to the primary lung and mediastinal targets achieved locoregional control but failed to address peritoneal metastases. Therapy combining gemcitabine, nedaplatin, anlotinib, and durvalumab was terminated prematurely due to grade 3 hepatotoxicity and pneumonia complications. The patient developed acute hypoxic respiratory failure, culminating in death 13 months post-diagnosis—nearly double the median OS for metastatic NMC (Fig.3).
3 Discussion
Primary pulmonary NMC represents a quintessential diagnostic challenge, characterized by a subtle onset—most commonly manifesting as a cough—and histomorphologic complexities that frequently lead to misdiagnosis [
12–
16]. Our comprehensive review of 86 published cases from 2011 to 2024 revealed an initial diagnostic error rate of 22% (19/86) [
3,
5,
7,
9,
13,
17–
45] (Fig.5), with misclassification as squamous cell carcinoma (SqCC) being the most prevalent due to overlapping immunohistochemical (IHC) markers (CK5/6
+, p40
+, TTF-1
–/Syn
–) (Fig.5). This diagnostic dilemma stems from the tumor’s cellular heterogeneity; while 78% of cases display squamous differentiation, 22% present as undifferentiated “small round blue cell tumors,” necessitating differentiation from neuroendocrine or sarcomatoid neoplasms [
17,
29,
46]. Established literature designates nuclear NUT expression via pathological IHC and FISH (using the C52 clone, with 100% specificity) as the diagnostic gold standard [
47–
50]. Nevertheless, the availability of FISH testing is not universal across hospitals, and financial concerns regarding FISH diagnostics are prevalent among patients. In scenarios where pathology reveals characteristic squamous epithelial cells with keratin pearl differentiation and IHC confirms positive NUT protein expression, a definitive diagnosis of NMC can still be made. Furthermore, NGS can identify NUT-related fusion genes, thereby providing additional diagnostic support. Previous case reports have consistently shown that no misdiagnoses occur in cases with typical pathology and positive immunohistochemical NUT protein expression. Moreover, NGS-based detection of NUT fusion genes not only aids in diagnosis but also facilitates the subtyping of NMC by delineating specific fusion genotypes, thereby enhancing our understanding and classification of this complex malignancy.
The lack of standardized therapeutic protocols for NMC is a testament to its aggressive nature and swift metastatic trajectory. Our comprehensive review (Table S1) demonstrated no significant survival advantage among various traditional treatment for advanced disease (
P = 0.175, Fig.5), with a median PFS of 2.1 months that aligns with historical benchmarks [
8].
However, in our cases, the integration of anti-angiogenic therapy at an early stage of treatment resulted in a longer PFS, providing patients with a greater opportunity to participate in clinical trials and offering them more potential treatment options, thereby extending their survival. We posit that the clinical benefits derived from this multimodal therapeutic strategy were primarily due to enhanced chemotherapy delivery facilitated by vascular normalization and increased radiosensitivity achieved through hypoxia reduction [
9,
51]. Furthermore, the blockade of VEGF played a crucial role in inhibiting peritoneal and hematogenous dissemination by downregulating CXCR4 and MMP9 [
52], thereby contributing to the improved outcomes observed in these patients. This approach underscores the potential of anti-angiogenic therapy in combination with traditional treatments to alter the course of this formidable disease.
Although phase II trials (ALTER-G-001, ETER701) have validated the efficacy of this approach in various malignancies, the evidence specific to NMC remains largely anecdotal. The contrast between the transient response observed in Jiang et al.’s study (50% tumor reduction) [23] and the sustained control in our cases underscores the pivotal role of toxicity management in achieving successful outcomes—balancing the need for dose modulation due to esophagitis or myelotoxicity with proactive supportive care within the treatment protocol.
The prevalence of the BRD4-NUTM1 fusion (87% of NMC, Fig.5) presents a therapeutic opportunity, as it renders NMC vulnerable to BETi by interfering with acetyl-lysine recognition in chromatin remodeling. Preclinical studies have shown that BETi downregulates oncogenes such as MYC, SOX2, and TP63, while HDAC inhibitors counteract EP300-driven acetylation [
53]. However, the limited response of BETi (NHWD-870) in our Case 1, despite the presence of the target, suggests potential resistance mediated by the tumor microenvironment or pharmacokinetic challenges in osseous metastases. Comprehensive preclinical research is essential to systematically unravel the pathobiological mechanisms driving NMC progression and to validate molecular targets for therapeutic intervention, with a particular focus on epigenetic dysregulation, oncogenicity of BRD4-NUT fusion, and tumor microenvironment interactions.
Ongoing trials (NCT05019716, NCT04247113) investigating CDK9 inhibitors and CAR-T therapies hold promise for further expanding the therapeutic arsenal. Our research findings position the integration of anti-angiogenic therapy in earlier treatment lines as promising strategy in this rare and aggressive malignancy.
In summary, this research effectively counters the prevailing therapeutic pessimism regarding primary pulmonary NMC by illustrating that strategically formulated multimodal treatments, which combine anti-angiogenic agents like anlotinib and bevacizumab with traditional therapies, can yield promising survival results. Our findings necessitate a transformative change in the sequence of treatments. We advocate for the upfront incorporation of anti-vascular therapy with platinum-doublet chemotherapy and prompt radiotherapy for stage IV disease, rather than relegating it to salvage scenarios. This strategy leverages the indicative “angiogenic switch,” a vascular-dependent phenomenon characteristic of the early metastatic progression in NMC, thereby optimizing treatment efficacy.
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