Neuregulin 1 (NRG1) fusion-positive high-grade spindle cell sarcoma: a distinct group of soft tissue tumors with metastatic potential

Abstract

Neuregulin 1 (NRG1) is an epidermal growth factor (EGF)-like ligand that activates receptor tyrosine kinases of the ErbB family of receptors. NRG1 gene fusions, which are rare (< 1%) but recurrent events in solid tumors, are an emerging oncogenic driver that is potentially actionable using ErbB-targeted tyrosine kinase inhibitors. Largely characterized only in carcinomas, we describe three cases of NRG1-rearranged sarcomas. The patients were all female, aged 32-47 years old. Two cases were deep-seated tumors in the lower extremities (right thigh and calf); one case presented as a uterine mass. The tumors measured 9-11.5 cm in greatest dimensions. Histologically, all three tumors were high-grade spindle cell sarcomas composed of monomorphic spindle cells arranged in interlacing fascicles. The tumor cells were set in loose collagenous stroma with branching, curvilinear thin-walled vasculature in the background. Cytologically, the neoplastic cells displayed ovoid to fusiform nuclei with fine stippled chromatin, inconspicuous nucleoli, scant to moderate clear to eosinophilic cytoplasm, occasional cytoplasmic vacuoles, and elongated cytoplasmic processes. Mitotic activity was elevated (> 20/10 high power fields) and tumor necrosis was present. None of the tumors expressed lineage-specific immunophenotypical markers. Targeted RNA-sequencing uncovered gene fusions involving NRG1 and the 5’ untranslated regions of PPHLN1, HMBOX1, or MTUS1. In all cases, the C-terminal EGF-like domain of NRG1 was preserved in the predicted chimeric protein product. All three patients developed metastatic disease within two years from initial presentation and were alive with disease at last follow-up (mean follow-up period = 19 months). In conclusion, we present the first case series of NRG1-rearranged sarcomas characterized by high-grade fascicular spindle cell morphology, non-specific immunoprofile and aggressive clinical behavior. Further studies are needed to determine whether this distinct subgroup of spindle cell sarcomas are amenable to targeted therapies.

Introduction

Neuregulins/heregulin (NRGs) comprise a family of epidermal growth factor (EGF)-like signaling molecules that interact with the ErbB family of transmembrane receptor tyrosine kinases (EGFR, ErbB2/HER2, ErbB3, and ErbB4). The NRG family of ligands have four members: NRG1, NRG2, NRG3, and NRG4. Receptor-ligand interaction induces homo- or heterodimerization of receptor monomers, which activates downstream signaling cascades and cellular responses including proliferation, migration, differentiation, and survival. Of the NRG proteins, NRG1 is well established to play important roles in development and human disease via the NRG1-ErbB2/ErbB3 signaling axis.^1–3

Over the past few years, NRG1 rearrangements have been detected in diverse solid tumors as a rare (< 1%) but recurrent oncogenic driver, including lung, breast, head and neck, pancreatic, prostate, renal, uterine, and ovarian cancers. Multiple NRG1 isoforms have been described. The main NRG1 isoform involved in NRG1 fusions belong to type III and contain a β-type EGF domain (β3 isoform).^4–10 The use of anti-ERBB2 and/or anti-ERBB3 inhibitors has been tested in preclinical models and clinical trials as a novel treatment paradigm in NRG1-rearranged cancers.^11–19 To date, NRG1 gene rearrangements have largely been reported only in carcinomas. We present the first case series of NRG1-rearranged sarcomas, describing the characteristic histopathologic features and clinical behavior of this novel mesenchymal tumor entity.

Materials and Methods

Study Cohort

Clinical data, including age, sex, and anatomic site were retrieved from pathology reports. Hematoxylin and eosin–stained slides from resection specimens were rereviewed. The study was approved by the Institutional Review Board.

Targeted RNA Sequencing

Detailed descriptions of MSK-Fusion, an amplicon-based targeted RNA NGS assay using the Archer™ FusionPlex™ standard protocol, were described previously.²⁰ Briefly, RNA is extracted from tumor formalin-fixed paraffin-embedded material followed by cDNA synthesis using the standard RNeasy FFPE Kit and protocol (Qiagen, Catalog #73504). A minimum of ten unstained slides and one hematoxylin and eosin–stained slide from formalin-fixed paraffin-embedded tissue (FFPE) were obtained for each sample and reviewed by a pathologist. Macrodissection was performed whenever indicated. Samples with at least 50 ng (200 ng preferred) of RNA were used for testing. cDNA libraries were made using the Archer™ FusionPlex™ standard protocol. Fusion unidirectional GSPs have been designed to target specific exons in 62 genes known to be involved in chromosomal rearrangements based on current literature. the final targeted amplicons are ready for 2×150bp sequencing on an Illumina MiSeq sequencer. FASTQ files are automatically generated using the MiSeq reporter software (Version 2.6.2.3) and analyzed using the Archer™ analysis software (Version 5.0.4). Each fusion call should be supported with a minimum of 5 unique reads and a minimum of 3 reads with unique start sites.

Results

Clinical summary

The clinical presentation and disease course are summarized in Table 1 and Figure 1. The patients were all females, aged 32, 25, and 47 years old at initial diagnosis. In two patients, the tumors were located at the lower extremities (right calf and left thigh) as large, deep-seated intramuscular masses, measuring 9.0 and 10.5 cm in greatest dimensions, respectively. One patient (case 3) received neoadjuvant chemotherapy (doxorubicin/ifosfamide) and radiation. Both tumors were surgically excised. Within two years (8 and 17 months), both patients developed distant metastases in the lungs. One patient (case 2) received adjuvant chemotherapy (doxorubicin/ifosfamide and anlotinib).

Table 1.

Patients Demographics, Pathologic Findings and Clinical Follow-up

Case	Age/Sex	Site (primary)	Greatest dimension (cm)	Site (metastases)	Time to progression (months)	Treatment	Status at last follow-up	Follow-up period (months)
1	47/F	Uterus	11.5	Peritoneum, Bowel	9	Surgical resection, radiation, chemotherapy (doxorubicin/ifosfamide), ERBB3 inhibitor	Alive with disease	22
2	35/F	Left thigh (lateral femoral muscle)	10.5	Lungs	8	Surgical resection, adjuvant chemotherapy (doxorubicin/ifosfamide, anlotinib) after metastasis developed	Alive with disease	10
3	32/F	Right calf	9	Lungs	17	Neoadjuvant chemotherapy (doxorubicin/ifosfamide) and radiation, surgical resection	Alive with disease	25

Figure 1.

Schematic illustrating disease course and clinical follow up of the cases 1-3. Abbreviations: CT: computed tomography; PET: positron emission tomography.

In the third patient (case 1), the patient presented with intermittent abdominal pain and underwent hysterectomy 3 months later, which revealed a 11.5 cm uterine mass with > 80% myometrial invasion. One month later, the patient developed local recurrence; she received chemotherapy (doxorubicin/ifosfamide) and later external beam radiation therapy. Her disease remained stable for 11 months until follow-up imaging showed recurrence of a large FDG-avid pelvic mass (Figure 2A–B) and multifocal intraabdominal metastases (omental and bowel implants). Three months thereafter, she was enrolled into a clinical trial (MCLA-128) for ERBB2/ERBB3 inhibitors. Subsequent computed tomography (CT) imaging demonstrated disease progression, and the patient transitioned to palliative treatment.

Figure 2. MTUS1-NRG1-fused sarcoma (Case 1).

A-B, Enlarging left pelvic mass (white arrows) superior to and encroaching the bladder seen in axial computed tomography (CT) (A) and sagittal magnetic resonance (MR) imaging (B) of the abdomen and pelvis. C, One of the many omental deposits showing tumor infiltrating adipose tissue (20X). D-E, The neoplastic cells are arranged in interlacing fascicles with relatively myxoid (D) and hypercellular (E) areas (100X). F, Cytologically, the cells exhibit clear cytoplasm, elongated eosinophilic cytoplasmic processes, hyperchromatic ovoid nuclei and inconspicuous nucleoli (200X). A-F, Hematoxylin and eosin stain.

At last follow-up, all three patients were alive with metastatic disease (median follow-up period of 19 months).

Histopathologic features

All three cases were cellular spindle cell tumors with a solid to multinodular growth pattern and appeared relatively circumscribed and unencapsulated on lower power, but could be seen infiltrating into surrounding tissue at higher magnification (Figure 2C, Figure 3A–B). The neoplastic cells were arranged in interlacing fascicles set in myxoid to pale pink collagenous stroma (Figure 2D–E, Figure 3B, Figure 4A–C). There was admixed, irregularly distributed, branching to curvilinear thin-walled vasculature in the background (Figure 3C, Figure 4B). Cytologically, the cells were monomorphic and characterized by hyperchromatic, ovoid to fusiform nuclei with fine stippled chromatin, inconspicuous nucleoli, scant to moderate amount of clear to eosinophilic cytoplasm with occasional vacuolization, and elongated cytoplasmic processes (Figure 2F, Figure 3D, Figure 4D). In the two resection cases, mitotic activity was brisk (> 20 per 10 high power fields), and occasional atypical mitotic forms were seen. Focal tumor necrosis (5%) was present. The histopathologic features are summarized in Table 2.

Figure 3. HMBOX1-NRG1-fused sarcoma (Case 2).

A, Resection from a 10.5 cm intramuscular tumor arising in the left thigh. The tumor exhibited a nodular growth pattern (10X). B, Tumor cells are arranged in fascicles and infiltrate surrounding mature skeletal muscle bundles (100X). C, Associated with the tumor cells are branching, thin-walled vasculature and collagenous stroma in the background (200X). D, Cytologically, the tumor cells display hyperchromatic ovoid to fusiform nuclei, moderate amount of eosinophilic cytoplasm, and elongated cytoplasmic processes. Mitotic figures are frequently seen (black arrows) (200X). A-D, hematoxylin and eosin stain.

Figure 4. PPHLN1-NRG1-fused sarcoma (Case 3).

A, Biopsy from a 9-cm right calf tumor revealed a cellular spindled tumor arranged in fascicles against a lightly myxoid stroma (200X). B, Cytologically, the tumor cells display monomorphic, round to ovoid nuclei with fine, stippled chromatin and inconspicuous nucleoli, scant amount of clear cytoplasm, and distinct cell membranes. Focally, thin-walled, linear to mildly dilated vasculature is present (200X). C, Wedge resection from the lung metastasis showing histomorphologic features similar to the primary (20X). D, Higher magnification of lung metastatic tumor demonstrating tumor cells arranged in fascicles and with ovoid to fusiform nuclei with fine stippled chromatin, occasional cytoplasmic vacuolization, and scattered mitotic activity. A-D, hematoxylin and eosin stain.

Table 2.

Microscopic, Immunohistochemical and Molecular Findings

Case	Histology	Cytomorphology	Mitotic activity (per 10 HPF)	Tumor necrosis (%)	Immunohistochemistry	Gene fusion partner 1	Gene fusion partner 2
1	Cellular, interlacing fascicles of spindle cells against myxoid to pale pink collagenous stroma; background thin-walled, curvilinear to branching vasculature	Monomorphic cells with scant to moderate clear to eosinophilic cytoplasm, occasional cytoplasmic vacuoles, elongated cytoplasmic processes, and hyperchromatic ovoid to fusiform nuclei with fine to stippled chromatin	> 50	5-10	(+) CD10, SMA (weak), ER (weak), PR (weak), cyclin D1 (focal) (−) desmin, myogenin, ALK1, CD117, WT1, HER2	MTUS1 exon 1 (5’UTR)	NRG1 exon 6
2			> 20	< 5	(+) SMA (rare), desmin (rare) (−) AE1/AE3, EMA, S100, SOX10, melan A, ERG, HER2	HMBOX1 exon 1 (5’UTR)	NRG1 exon 2
3			2 (core biopsy)	None (core biopsy)	(+) CD99, BCL2 (−) PanCK, EMA, S100, SOX10, HMB45, melan A, MiTF, SMA, desmin, myogenin, ERG, CD31, CD34, CD68, CD45, synaptophysin, chromogranin, STAT6	PPHLN1 exon 1 (5’UTR)	NRG1 exon 2

HPF: high power fields

Immunohistochemical summary

All three cases showed a nonspecific and essentially “null” immunophenotype, with focal and weak SMA, and were negative markers of epithelial, Schwannian, melanocytic, muscular or vascular differentiation (Table 2). A HER2 immunohistochemical stain performed on case 1 and 2 was negative.

NRG1 fusion transcript

A targeted RNA sequencing fusion panel (Archer) confirmed in-frame gene fusion transcripts between exon 1 of PPHLN1 [NM_016488] (case 1), HMBOX1 [NM_001135726] (case 2) and MTUS1 [NM_001363065] (case 3) and exons 2 or 6 of NRG1 [NM_013958] (Table 2). In all three cases, the 5’ untranslated regions of the partners were fused to NRG1, of which the C-terminal EGF-like domain was preserved in the predicted chimeric protein product (Figure 5).

Figure 5.

Schematic of predicted fusion proteins encoded by NRG1 gene fusions. The fusions involve exons 1, which encode for the 5’ UTR, of PPHLN1 [NM_016488], HMBOX1 [NM_001135726] and MTUS1 [NM_001363065] and either exon 2 or exon 6 of NRG1 [NM_013958]. The chimeric protein products are predicted to be in-frame and include the EGF-like domain (178-222 aa) in NRG1 (β3 isoform, 241 aa) for all three cases. Vertical dotted lines represent exon boundaries. Abbreviations: 5’UTR, 5’ untranslated region; aa, amino acids; EGF: epidermal growth factor; Ig, immunoglobulin-like domain.

Discussion

NRG1 is encoded by the NRG1 gene, which located on chromosome 8p12. The NRG1 gene is translated to multiple different isoforms. NRG1 proteins are structurally related to EGF and contain an EGF-like domain that binds and activates ErbB3 and ErbB4. Upon binding by NRG1, ErbB3 and ErbB4 undergo homo- or heterodimerization with ErbB members, including ErbB2, which leads to activation of downstream signaling, including PI3K-AKT and MAPK pathways that regulate oncogenic cellular processes (e.g., cell proliferation, survival, migration, etc).^1,2 During development, the neuregulin/ErbB signaling network regulates the assembly of neural circuitry and myelination of the central and peripheral nervous system.²¹ Interestingly, this pathway has been shown to promote schwannoma tumorigenesis and the proliferation of neoplastic Schwann cells in human malignant peripheral nerve sheath tumors (MPNST).^22,23 Moreover, constitutive activation of neuregulin/ErbB signaling was identified in clear cell sarcoma, a malignant mesenchymal tumor with melanocytic differentiation.²⁴ Whether or not activation of neuregulin/ErbB signaling across reflect similarities underlying tumorigenesis of these distinct entities remain to be further investigated.

NRG1 gene fusions are rare across different types of cancer and are detected in < 1% of solid tumors in most large series.^3,4 Initially discovered in 2014 as CD74-NRG1 in lung adenocarcinoma,⁵ NRG1 fusions with many other partners have been discovered since then in multiple tumor types, including breast, head and neck, colorectal, pancreatic, biliary tract, ovarian, uterine, renal cancers.^3–10 In one study that profiled > 20,000 specimens across multiple solid tumors, 41 (0.2%) harbored NRG1 fusions, and one sarcoma was detected. However, no additional detail regarding this case was provided.⁴ Many of these studies showed that the NRG III-β3 isoform is involved in the fusion. To our knowledge, the partners identified in the NRG1-rearranged sarcomas in the current study have not been reported previously. It has been postulated that the fusion confers protumorigenic properties via overexpression of NRG1 by placing it under control of an alternative promoter or transcription regulator; alternatively, NRG1 fusion causes loss or alteration of protease cleavage sites, resulting in excess NRG1 tethered to the cell surface, which in turns promotes uncontrolled activation of ErbB-PI3K/MAPK downstream signaling.³

Since NRG1 proteins are ligands of ErbB receptors, there is intense interest in the use of ErbB-targeted treatments, such as monoclonal antibodies and small molecular tyrosine kinase inhibitors (TKIs), in NRG1-rearranged cancers, with efficacy demonstrated in preclinical models.^11–15 Partial responses have been reported in patients with invasive mucinous adenocarcinomas of the lungs and pancreatic ductal adenocarcinomas.^16–19 In contrast, a few case reports have shown progressive disease following afatinib or anti-ErbB3 antibody treatment.³ This illustrates the need to recruit a larger number of patients with NRG1 fusion-driven cancers to evaluate the efficacy of ErbB-targeted treatments.

The differential diagnosis NRG1-rearranged sarcomas is broad and includes other high-grade spindle cell sarcomas. Synovial sarcomas commonly present as deep-seated tumors in soft tissue or visceral organs that can metastasize to distant organs, and the monophasic variant is characterized by monomorphic spindle cells with closely apposed nuclei arranged in long intersecting fascicles. However, synovial sarcomas often harbor epithelial differentiation and are characterized by the presence of SS18 gene rearrangements.^25,26 MPNST is another high-grade spindle cell sarcoma that enters the differential diagnosis. Also characterized by cellular fascicles of spindle cells, the diagnosis of MPNST is best rendered in a patient with a history of neurofibromatosis 1, or a high-grade tumor arising from a preexisting benign nerve sheath tumor or in association with a peripheral nerve.²⁷ The loss of H3K27me3, when present, could be a useful adjunct to diagnose MPNST.²⁸ In the female genital tract, endometrial stromal sarcomas (ESS) are in the differential diagnosis of NRG1-rearranged sarcomas. The presence of CD10 or cyclin D1 is nonspecific. Detection of characterize gene fusions, such as JAZF1-SUZ12 for low-grade ESS,²⁹ BCOR fusions/internal tandem duplication and YWHAE rearrangements for high-grade ESS,^30–32 helps establish the diagnosis.

A major limitation of this study is the small sample size and the limited clinical response data to ERBB2/ERBB3 inhibitors. Despite the limited number of patients, all three cases presented in young adult women, had overlapping morphology and followed a highly aggressive course with developing metastatic disease. In the single case where the patient did enroll in an ERBB2/ERBB3 inhibitor clinical trial, her disease progressed and she withdrew after 2 months. However, at the time of enrollment, the patient disease had already advanced disease for almost 2 years, having experienced multiple disease recurrence and metastases on conventional chemotherapy. Therefore, it remains unclear how NRG1-rearranged sarcomas may respond to ERBB2/ERBB3 inhibitors. Unlike carcinomas, targeted therapies are still very limited and urgently sought after in the treatment of mesenchymal tumors. Examples include the use of KIT/PDGFRA inhibitors in the treatment of gastrointestinal stromal tumors,³³ anaplastic lymphoma kinase (ALK) inhibitors in inflammatory myofibroblastic tumors,³⁴ tropomyosin receptor kinase (TRK) inhibitors in neurotrophic tyrosine receptor kinase (NTRK)-rearranged soft tissue tumors,³⁵ and denosumab in giant cell tumor of bone.³⁶ Larger scale molecularly-driven clinical studies are needed to determine whether ERBB2/ERBB3 inhibitor is a viable therapeutic option in NRG1-rearranged sarcomas.

In conclusion, we report the first case series of NRG1-rearranged sarcomas. Histopathologically, these are high-grade spindle cell sarcomas characterized by fascicular growth of monomorphic spindle cells. NRG1-rearranged sarcomas appear to follow an aggressive clinical course and lack substantial response to conventional chemotherapy. Given the established role of NRG1 in the activation of ERBB2/ERBB3 signaling, recognition of NRG1 gene fusions in sarcomas may offer a targetable therapeutic strategy with the use of ERBB2/ERBB3-specific TKIs or antibodies.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.