NOTCH3 missense mutations as predictor of long-term response to gemcitabine in a patient with epithelioid hemangioendothelioma

Purpose Epithelioid hemangioendothelioma (EHE) as a very rare malignant vascular tumor belongs to the heterogenous group of soft-tissue sarcomas. Depending on the clinical course of the disease, interdisciplinary treatment concepts are required, including surgery, radiotherapy and systemic cancer therapy. However, due to its uncommonness, standard treatment options are lacking so far, especially in advanced disease with distant metastases. Methods and results Here we report on an unusual case of a patient with metastasized EHE showing long-term response to second line treatment with gemcitabine over almost 2 decades. Cancer genome sequencing of the patient’s tumor tissue detected a NOTCH3 missense mutation which could provide an explanation for these clinical findings. NOTCH3 is known to be a mediator of resistance towards gemcitabine-based cancer treatment, at least in pancreatic cancer and non-small cell lung cancer. Conclusion The observation that this missense mutation of NOTCH3 is associated with an increased response to treatment with gemcitabine in EHE can be used prospectively to assess NOTCH3 as potential biomarker for predicting therapy response to gemcitabine.

Epithelioid hemangioendothelioma (EHE) represents a rare entity of soft-tissue sarcoma (STS) with a prevalence of < 1/1,000,000, mainly affecting young and middle-aged adults (de Pinieux et al. 2021;Flucke et al. 2014;Guo et al. 2017;Lau et al. 2011;Rosenbaum et al. 2020;Stacchiotti et al. 2021a, b). As a malignant vascular tumor, the disease originates from endothelial or pre-endothelial cells and can manifest in any part of the body, with the lung being the most frequent location (30% of all cases) (Lau et al. 2011;Sardaro et al. 2014). The clinical course varies from low malignant to highly aggressive disease with the tendency of rapid distant metastasis (Stacchiotti et al. 2021a, b). The diagnosis is often made incidentally during routine imaging. However, depending on the site of manifestation, patients may also be alerted to the disease by general symptoms such as weight loss and anemia, respiratory symptoms, bone pain or neurologic abnormalities which are associated with a worse prognosis (Amin et al. 2006;Bagan et al. 2006;Kitaichi et al. 1998). Histologically, EHE is defined via accumulation of epithelioid endothelial tumor cells embedded in a myxohyaline or fibrous stroma (Fig. 1a). Of note, diagnosis 1 3 of EHE can be challenging as it shares common histopathological features of several carcinomas, sarcomas (especially epithelioid and angiosarcoma) as well as mesothelioma or even autoimmune diseases like sarcoidosis (Anderson et al. 2015;Rosenberg and Agulnik 2018;Stacchiotti et al. 2021a, b).
A common genetic alteration of EHE is the occurrence of specific gene fusions, including WWTR1-CAMTA1, being the most frequently detected translocation in EHE, or YAP1-TFE3 (Antonescu et al. 2013;Errani et al. 2011). These genetic aberrations have been discussed to contribute to pathophysiology of EHE since both WWTR1 and YAP1 represent central effector proteins of the Hippo pathway mediating pro-oncogenic transcriptional effects (Merritt et al. 2021;Salem and Hansen 2019;Wu and Yang 2018;Zender et al. 2006).
For patients with limited disease, tumor resection shows curation rates of 70 up to 80% (Rosenbaum et al. 2020;Stacchiotti et al. 2021a, b). In metastatic disease, however, no standard therapy is established so far and treatment options are still very limited. Retrospective analyses investigating several different chemotherapeutic agents, small molecule inhibitors and immunomodulatory agents showed only limited response rates (Sardaro et al. 2014). Exemplarily, a retrospective international case series showed a progression-free survival of no more than 5 months and no partial response in a cohort of patients with advanced EHE under systemic treatment with doxorubicin and ifosfamide (Frezza et al. 2021).
Here, we present an unusual and rare case of a metastasized EHE with a long-term response over 205 months (17 years) to a second line systemic treatment with gemcitabine after initial tumor resection. Our case refers to a casuistic of EHE partially displayed in a previous report by Pintoffl et al. (Pintoffl et al. 2009). At this time point, the authors described the unique course of this case under systemic treatment with gemcitabine over a period of 72 months. Referring to this, almost 14 years later, we provide an update on this very unusual case and relate the therapeutic response to meanwhile available molecular features of the tumor tissue.
In June 2000, a 30-year-old man was diagnosed with EHE of the right scapula. After computed tomography (CT) scan-based exclusion of distant metastases a complete tumor excision was performed by means of a Tikhoff-Linberg procedure type IV (Voggenreiter et al. 1999). In November 2001, a subsequent CT scan showed multiple lung, liver and bone metastases (Fig. 1b). Between November 2001 and February 2002, the patient underwent both local radiotherapy of multiple osteolytic lesions and systemic treatment with doxorubicin (25 mg/m 2 ) in combination with ifosfamide (2 g/m 2 ), each compound applied for three consecutive days, given for four therapy cycles with a cycle length of 21 days, respectively, in total.
After a subsequent disease progression in March 2002, a systemic second-line treatment with gemcitabine (administered with 1 g/m 2 on days 1, 8 and 15 of each therapy cycle comprising a total of 28 days) was initiated. Between March 2002 and January 2008, the patient received in total 62 therapy cycles leading to a stabilization of the disease with even partial regression of the pulmonary lesions. Subsequently, treatment with gemcitabine was discontinued. In June 2009, a general tumor progression was observed. As a result, a stereotactic irradiation of two unstable lumbar osteolytic lesions as well as of newly detected cerebral metastases was combined with a systemic treatment, again employing gemcitabine. Gemcitabine treatment was consequently administered for a total of 164 therapy cycles until January 2021. Remarkably, in that time period no further tumor progression was observed. The patient showed an excellent overall tolerability without any signs of long-term toxicities enabling an unrestricted participation in everyday life (ECOG performance status grade 0). The patient was fully employed and physically active under systemic treatment with gemcitabine and had no need of supportive medication. In February 2021, detection of further systemic tumor progression led to treatment change to pazopanib (Fig. 1c).
The present case highlights the potential benefit of a systemic treatment with gemcitabine in EHE. Gemcitabine as an anti-metabolite mediates its cytotoxic effects via integration into the DNA of tumor cells leading to an abortion of DNA synthesis (Plunkett et al. 1995). To our knowledge, only case series of patients with advanced EHE treated with gemcitabine, mostly in combination with docetaxel, are reported (Frezza et al. 2021;Sabile et al. 2021;Zhou et al. 2020). According to these publications, systemic treatment of EHE with gemcitabine, at least in combination with docetaxel, could lead to a stabilization of the disease in some patients. However, long-term response to therapy with single treatment of gemcitabine is rarely described so far. Furthermore, in most cases, an assessment of the genetic profile of the tumor has not been performed.
Our patient presented a YAP1-TFE3 fusion which has been described to act as tumor-driving gene fusion in a subset of EHE (Antonescu et al. 2013;Rosenbaum et al. 2020;Szulzewsky et al. 2021). Previous studies revealed the significance of YAP1 and TFE3 for carcinogenesis in several tumor entities, such as liver cancer, breast cancer, prostate cancer or renal cancer (Salem and Hansen 2019;Wu and Yang 2018;Yin et al. 2019;Zender et al. 2006). Of note, overactivation of Hippo/YAP1 signaling is known to be associated with resistance to anti-metabolites, like gemcitabine, at least in pancreatic and colorectal cancer (Nguyen and Yi 2019). However, the impact of YAP1-TFE3 fusion on response to gemcitabine in EHE is not investigated so far.
Interestingly, further somatic molecular genetic analyses of a pelvic bone metastasis (Ki-67 proliferation index of 5%) in 2021 by means of the Illumina HiSeq/NovaSeq system and a 750-gene next-generation sequencing panel identified somatic mutations of PTK7, RPTOR as well as a heterozygous NOTCH3 missense mutation (p.Asn1964Ser). To our knowledge, the significance of this NOTCH3 missense mutation has not been described in the literature. However, protein domain analyses revealed that the abovementioned missense mutation affects ankyrin repeat domains of NOTCH3 that are crucially involved in mediating protein-protein interactions and alterations in these domains cause functional impairment and lead to disease (Li et al. 2006). It could be shown that NOTCH3 is involved in mediating resistance to gemcitabine in pancreatic and non-small cell lung cancer (Hu et al. 2018;Xiu et al. 2021). In vitro studies revealed that NOTCH3 promotes resistance to gemcitabine via activation of PI3K/Akt signaling in pancreatic cancer (Yao and Qian 2010). Additionally, NOTCH3 could be identified as parameter for predicting therapy response to gemcitabine in pancreatic cancer: expression of NOTCH3 inversely correlated with therapy response and overall survival of patients treated with gemcitabine (Eto et al. 2013). As a result, siRNAs or γ-secretase inhibitors targeting NOTCH3 are currently under investigation to overcome therapy resistance and sensitize pancreatic and non-small cell lung cancer to treatment with gemcitabine (Hu et al. 2018;Yao and Qian 2010). The role of NOTCH3 in mediating chemoresistance in EHE remains unclear so far.
The clinical findings presented in this case provide first evidence for a relevance of NOTCH3 in the context of gemcitabine-based treatment of EHE. Our case highlights the potential benefit of a monotherapy with gemcitabine in advanced EHE with a tolerable toxicity profile. Since genetic analyses from EHE patients previously treated with gemcitabine are lacking so far, we here show a potential correlation between the therapy response to gemcitabine and the presence of a NOTCH3 missense mutation. Further prospective clinical trials are needed to investigate the role of gemcitabine as systemic treatment option in a larger cohort of patients with advanced EHE and to further assess NOTCH3 as potential biomarker for predicting therapy response to gemcitabine.
Author contributions MH and CH conceived and designed the study. MS, SM, SS, MS, SB, PK, UML and LZ conducted the patient data, as well as the medical evaluation and analysis. MS, MH and CH analyzed the data. MS, MH and CH prepared the figure; MS, MH and CH wrote the first draft of the manuscript. LZ and CH contributed to the data interpretation and manuscript edit. All of the authors critically reviewed, read, and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.
Funding Open Access funding enabled and organized by Projekt DEAL. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy-EXC 2180-390900677.

Data availability
The data supporting our findings are available from the corresponding author upon reasonable request.

Conflict of interest
The authors declare no competing financial interests.
Informed consent After obtaining written informed consent from the patient, this case study was performed in accordance with the Declaration of Helsinki.
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