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Tumor regression on sequential contrast enhanced CT scans (CECT) of the chest. (A) Axial chest CECT (performed on day 0, i.e. before the onset of the multimodal NUT therapy regimen) - large, highly attenuated lung mass occupying the entire right upper lung lobe with invasion of the mediastinum as well as tumoral spread to the hilar and pre-tracheal lymph nodes. (B) First follow-up (FU) (day 20) - increasingly heterogeneous tumor attenuation and slight shrinkage. (C) Second FU (day 55) - further loss in tumor attenuation (vascular supply) as well as strong volume reduction on chest CECT. (D) Third FU (day 77) - stable situation with respect to the residual tumor manifestations in the right lung and mediastinum. (E) Axial chest CECT (day 0) - right sided pleural metastases and epiphrenic tumor mass. (F) First FU (day 20) - volume reduction of the pleural and epiphrenic tumor manifestations. (G) Second FU (day 55) - stable situation. (H) Third FU (day 77) - slowly progredient epiphrenic and pleural tumor with increased marginal attenuation.
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The CT scan revealed a large pulmonary mass (12 x 11.8 x 11 cm) in the right upper lobe with compression of the right upper lobe artery and bronchus, right-sided malignant pleural effusion with pleural metastases, enlarged ipsilateral mediastinal lymph nodes and a mediastinal shift to the left side ( Figure 1A ). Reference pathology performed at the University of Erlangen, Germany, reported an undifferentiated highly malignant tumor with spindle and polygonal cells and diffuse expression of p63 and pancytokeratin, which supported the diagnosis of a non-keratinizing SCC ( Figure S1 ). The final diagnosis of NC was made via immunostaining with an anti-NUT antibody showing a strong nuclear signal and an additional fusion panel RNA sequencing, which revealed a BRD3::NUTM1 gene fusion ( Figure 2  and  Figure S1 ). S1 ). Staining for PD-L1 showed a TPS (tumor proportion score) and CPS (combined positive score) <1% ( Figure S1 ). Hospital of Tubingen, a German Comprehensive Cancer Center specialized in NC tumors, for further treatment. Here, an immediate CT scan displayed further progressive disease (PD) with additional involvement of the pleura, the diaphragm, the pericardium as well as mediastinal lymph nodes ( Figure 1E ). A CT scan after the second therapy cycle showed a partial response (PR) of the primary tumor and of pleural and pulmonal metastases as well as shrinking mediastinal lymph nodes ( Figure 1G ). A follow-up CT scan demonstrated a generally stable disease (SD), but with a slowly progressive epiphrenic tumor mass ( Figure 1 ). T-VEC injection was administered into a highly vital right epiphrenic tumor mass, because a previous CT-Scan had revealed a necrotic primary tumor in the right upper lobe of the lung ( Figure 1D ), though slowly progressive right epiphrenic tumor manifestations ( Figure 1 ). This resulted again in high serum viral loads as detected by quantitative real-time PCR for HSV-1 DNA (HSV1&2,
BRD3::NUTM1 gene translocation being found in fusion RNA panel sequencing of tumor tissue. (A) TruSight RNA fusion panel sequencing (Illumina) revealed BRD3 as NUTM1 fusion partner. Integrated genome viewer split-screen view of the breakpoint regions on chromosomes 9 (left) and 15 (right). Read alignments of paired-end RNA sequencing of the identified BRD3::NUTM1 fusion event are shown. Mate pairs mapped to the fusion reads in the BRD3 (purple color) and NUTM1 (green color) locus are shown. (B) Estimated BRD3::NUT fusion protein with breakpoints after coding exon 8 of BRD3 (arrow; chr9:136,905,154, amino acid G549) and before exon 4 of NUTM1 (chr15:34,645,933), resulting in the respective fusion oncoprotein. Interestingly, the extraterminal domain (BRD3, amino acids 564-641) and the second nuclear localization sequence coded on exon 9 of BRD3 are not contained in the fusion oncoprotein.
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The final diagnosis of NC was made via immunostaining with an anti-NUT antibody showing a strong nuclear signal and an additional fusion panel RNA sequencing, which revealed a BRD3::NUTM1 gene fusion ( Figure 2  and  Figure S1 ).
Multimodal NUT carcinoma (NC) therapy regimen, including Immunovirotherapy as a novel add-on therapy. Multimodal treatment plan. Days since first admission at specialized NC Cancer Center at University Hospital Tuebingen are shown. Mutlimodal treatment was performed with three-weekly cycles of T-VEC followed by pembrolizumab and accompanied by chemotherapy. Immunovirotherapy: T-VEC (108 PFU) blue arrwos, pembrolizumab (200 mg) green arrows. Chemotherapy: CTx #1 Carboplatin/Paclitaxel (performed before referral to UKT), CTx #2 Ifosfamide/Etoposide, CTx #3 Vincristin/Cyclophosphamid/Doxorubicin/Etoposide 75%, CTx #4/#5 Vincristin/Cyclophosphamid/Doxorubicin/Etoposide 50%. Radiation: 11 x 3 Gy = 33 Gy. HDAC inhibitor: oral suberoylanilide hydroxamic acid (SAHA). PD: progressive disease, SD: stable disease, PR: partial response according to RECIST 1.1.
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Shortly after admission, an intensive multimodal therapy was initiated ( Figure 3 ). T-VEC (108 PFU/ml) was administered intratumorally under ultrasound guidance on day six after the first admission ( Figure 3 ,  4D ).
Evidence for highly efficient replication of T-VEC in NC tumor cells. (A) Serum viral loads over time determined by standard HSV-1 DNA real-time PCR from patient blood samples (blue arrows indicate time points of intratumoral (i.t.) injections of T-VEC). The first three times, T-VEC was administered into the right pulmonary mass identified as the primary tumor. At the time of the third i.t. injection of T-VEC, CT revealed necrosis of this pulmonary mass; as a consequence, the third injection applied again to this area no longer resulted in the production of relevant amounts of T-VEC DNA (days 56-77). Hence, injection site was changed for the fourth application of T-VEC to a highly vital and progressive epiphrenic tumor mass, now resulting again in the production of relatively high loads of HSV-1 DNA. (B) Serum LDH serves as a surrogate proliferation marker for NC (high levels are supposed to indicate a highly proliferative state). Each treatment cycle of the multimodal therapy regimen led to the reduction of serum LDH values below the upper normal level of 250 U/l after 5 to 10 days. However, shortly after completion of each cycle, LDH levels increased again in a highly regular manner. (C) Agarose gel electrophoresis of a PCR from serum samples employing T-VEC specific primers. Upper panel: Band intensities correlated well with results of standard HSV-1 qPCR (A). Highest intensities were found after the first (d7-d12) and fourth (d85) T-VEC administration. Lower panel: A schematic overview of the T-VEC genome; primers (purple) bind to the flanking regions of the hGM-CSF transgene (blue) located within the terminal repeats (RL) of the unique long region (UL), being highly specific for the T-VEC genome. (D) The first three doses of T-VEC were administered into a tumor mass in the right upper pulmonary lobe of the lung via dorsal fine needle injection in the scapular line, whereas the fourth dose was injected into a right sided highly vital epiphrenic tumor manifestation at the midaxillary line. Created with BioRender.com.
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T-VEC (108 PFU/ml) was administered intratumorally under ultrasound guidance on day six after the first admission ( Figure 3 ,  4D ). VZV R-GENE  Kit, Biomerieux) comparable with the level obtained after the first T-VEC injection in the initial right pulmonary tumor mass ( Figure 4 ). Virotherapy was again accompanied by chemotherapy (VCDE; 50%) and pembrolizumab.