GLI1-altered Mesenchymal Tumors of the Head and Neck

Abstract

GLI1-altered mesenchymal tumors are a group of soft tissue tumors that have emerged as a new entity, in large part, as a result of increased next-generation sequencing (NGS)-based DNA and RNA sequencing of tumors. Over the last decade, multiple case reports and small series have reported such tumors involving multiple sites (including soft tissue, head and neck and viscera). They appear to have rather distinctive morphology and can either show GLI1-gene fusions or GLI1-gene amplifications. Although cases with metastasis are increasingly recognized, definite morphologic indicators of metastasis have not yet been identified. In this review, we focus on GLI1-altered mesenchymal tumors of the head and neck, with an emphasis on clinicopathologic features, molecular findings and approach to the diagnosis.

INTRODUCTION

The advent of next-generation sequencing (NGS) has heralded the identification of entities with specific genetic abnormalities in the form of gene fusions and / or gene mutations. One such emerging entity is the GLI1-altered mesenchymal tumor, an umbrella term proposed for a group of neoplasms with similar morphology and characterized by either gene fusions or gene amplifications of the GLI1 gene. Over the last decade, multiple studies ^1–15 have reported a small series of cases with GLI1 alterations involving various sites. In this review, we focus on the GLI1-altered mesenchymal tumors of the head and neck.

Evolution of the Entity

Tumors with alterations of GLI1 (GLI family zinc finger 1 or glioma-associated oncogene homologue 1) were first reported by Dahlen et al. ¹⁶ in 2004 in a study of 5 tumors that showed t(7;12)(p21–22;q13–15) translocation resulting in fusion of the beta-actin gene with GLI1 (ACTB::GLI1). These 5 tumors, located in the calf, stomach and tongue, showed rather similar morphology of multilobulated, infiltrative growth with the tumor composed of uniform spindle-shaped cells with small amounts of eosinophilic cytoplasm and nuclei with vesicular chromatin. The tumor cells were arranged around small thin-walled arborizing vessels and showed no cytologic atypia or pleomorphism. In 2 cases, tumor cells showed subendothelial protrusion into the vascular lumina (reminiscent of a pericytic neoplasm). Given the morphology the authors coined the term "Pericytoma with t(7;12)”. Immunohistochemical stains for smooth muscle actin and laminin are reportedly positive in these cases.

Since this original description, numerous studies have reported GLI1 fusion positive tumors with various gene partners including ACTB, MALAT1, PTCH1, APOD, DERA, SYT, NCOR2, HNRNPA1, NEAT, TXNIP and PAMR1 ^1–14; and at various anatomic sites including head and neck, bone, retroperitoneum, skin and subcutis, and other visceral organs involving gastrointestinal, genitourinary, and gynecologic tracts.

GLI1 alterations have also been reported in two other morphologically distinctive mesenchymal neoplasms involving the gastric wall, plexiform fibromyxoma and gastroblastoma. ^{1, 12, 17, 18} Both of these entities are associated with MALAT1::GLI1 gene fusion and show morphologies distinct from the other GLI1-altered mesenchymal tumors. Plexiform fibromyxoma is a benign neoplasm that occurs in the gastric wall with a multinodular/plexiform pattern of growth. Morphologically, this hypocellular spindle cell neoplasm is composed of bland spindle cells in a predominantly myxoid to collagenous stroma. The lesional cells are positive for SMA. ^{12, 17} Gastroblastoma also occurs in the gastric wall and shows a biphasic spindled to epithelioid morphology, with the nested epithelial cells staining for pan-cytokeratin. ^{1, 18}

The molecular alterations of GLI1 associated tumors was expanded in a study by Agaram et al. in 2019 ¹⁹ when they reported 10 cases of mesenchymal neoplasms that were similar to the GLI1 fusion positive reported cases, but instead showed GLI1 gene amplification. These cases were located in soft tissues of the limbs, trunk, and head and neck. Co-amplification of CDK4 and MDM2 genes were also identified in 80 to 90% of the of the cases. Additional studies have also reported subsequently on GLI1 amplified mesenchymal tumors. ^{5–9, 13, 14}

Although initially reported under different terminologies including pericytoma, glomoid neoplasms, etc. the term "GLI1-altered mesenchymal tumor” is the currently proposed terminology to include cases with GLI1 gene fusions as well as GLI1 gene amplifications. ^{15, 20}

Clinicopathologic features

GLI1-altered mesenchymal tumors usually occur in adults with a median age of 40 to 46 years, with a wide age range of 1 to 80 years. No sex predilection is noted. The size of the tumors in soft tissues ranges from less than 1 cm up to 20 cm, with a median size of 2 to 4 cm. Extremities and head and neck locations appear to be the most common sites, involving half of the reported cases. The rest of the sites include intra-abdominal, trunk, pelvis, intrathoracic and other locations.

Morphologically, the tumors are typically well-circumscribed and show a multinodular, lobulated growth pattern with lobules of tumor cells separated by fibrous bands. Tumors are usually composed of monomorphic, ovoid to spindle cells arranged in sheets or small nests, with the nests showing intervening dense vascularity in the form of capillary network. In some foci, the tumor cells also appear to protrude into the vascular spaces, although lined by a layer of endothelial cells. (Figure 1) Nuclear atypia or pleomorphism is absent, although rare cases with atypia and pleomorphism have been reported. ²¹ Mitotic activity is typically low ranging from 1–2 per 10 high-power fields. Tumor necrosis is uncommon.

Figure 1:

Morphology of GLI1-fusion positive mesenchymal tumor. A. Low power H&E image showing involvement of the tongue submucosa by a cellular lesion. (100x) B. High power H&E image showing small uniform cells in a nested pattern with intervening thin fibrous septa and rich vascularity. (400x). C. Immunohistochemical stain for S100 showing patchy positivity in the tumor cells. (200x) D. FISH image (Green – GLI1, Red – DDIT3) showing evidence of rearrangement of the GLI1 gene.

A few unusual morphologic patterns viz. cytoplasmic vacuolization, spindling and fascicular arrangement, cells with granular cytoplasm, rosetting, microcystic growth, high grade sarcomatoid morphology and extensive myxoid change have been reported in some cases. ¹³(Figure 2)

Figure 2:

Unusual morphologic features of GLI1 altered mesenchymal tumors. A. H&E image showing areas of cytoplasmic vacuolization. (400x) B. H&E image showing extensive myxoid change in the tumor. (200x) C. H&E image showing biphasic pattern with areas on the left showing conventional morphology while area on the right shows spindled fascicular pattern. (200x) D. H&E image showing nodular areas separated by thick fibrous septa. (100x)

Immunohistochemically, although initially reported to be positive for SMA, studies to date have noted that the immunoprofile is relatively nonspecific. Tumors have been shown to be positive for CD56, SMA, and S100 in varying proportions. Other markers such as desmin, CD31, ERG, myogenin, MyoD1, pan-cytokeratin, GFAP, calponin, etc. are negative.

The subset of GLI1 amplified mesenchymal tumors show consistent overexpression of CDK4, MDM2 and, on some occasions, STAT6. Therefore, the immunohistochemical expression of these markers may aid in the identification of GLI1 amplified tumors. ¹⁹ GLI1 antibody has also recently been shown to be useful in the identification of GLI1 altered tumors. ¹⁰ The reported sensitivity and specificity for the GLI1 antibody is 91% and 98%, respectively.

Molecular features

GLI1, located on chromosome 12q13.3, encodes a transcription factor of the Kruppel family of zinc finger proteins, which plays a critical role in the Hedgehog signaling pathway. GLI1 gene fusions and amplifications lead to an increase in the expression of both GLI1 mRNA transcripts and protein, and subsequent activation of downstream targets with key roles in tumorigenesis. ^{19, 21}

GLI1 fusions operate through a mechanism known as promoter swapping, in which the promoter region of GLI1 is substituted with the highly active promoter region of the 5’ fusion partner. This leads to abnormal mRNA and protein production. GLI1 fusions typically occur within exons 4, 5, 6, or 7. These breakpoints lead to the retention of the functional FOXP coiled-coil and DNA binding zinc finger domains encoded by the 3’ portion of GLI1. Despite being truncated, the resulting protein retains its ability to activate downstream targets. ^{20, 21}

GLI1 amplification was first reported in gliomas in the 1990s resulting in the gene being designated ‘glioma-associated oncogene 1’. ^{22, 23} GLI1 amplification is most frequently seen in soft tissue sarcomas (6%) followed by glioma (4%), melanoma (2%), and lung adenocarcinoma (1%). ¹⁹ Among the soft tissue and bone sarcomas with GLI1 amplification, liposarcomas (well-differentiated/dedifferentiated) are the most common (85%), followed by intimal sarcoma (5%), parosteal osteosarcoma (5%) and tumors classified as sarcoma, not otherwise specified (5%).¹⁹

GLI1 is located on 12q13.3, less than 100 kilobases (KBs) from DDIT3, and 300 KBs from CDK4 (12q14.1) and STAT6 (12q13.3). MDM2 is located at 12q15, about 11 Mb from the GLI1/CDK4 gene cluster. (Figure 3) As the GLI1 gene is located within an oncogene-rich region, the 12q13–15 locus, the amplicon is composed of GLI1, DDIT3, CDK4, and MDM2. In the study by Agaram et al. ¹⁹, DDIT3, located adjacent to GLI1, showed amplification by fluorescence in-situ hybridization (FISH) in all of the tested cases, and was proposed as a surrogate test for GLI1 gene fusions and gene amplifications. Co-amplification of CDK4 and MDM2 is the defining genetic event in several bone and soft tissue tumors, such as well differentiated/dedifferentiated liposarcoma, intimal sarcoma, low-grade central osteosarcoma, and parosteal (juxtacortical) osteosarcoma. ²⁴ In a recent study, custom FISH probes for GLI1 gene have been used to detect GLI1 gene amplification or rearrangement. ⁵ More recently, commercial GLI1 FISH probes are available and could be used for analyzing GLI1 gene alterations.

Figure 3:

Schematic representation of the close proximity of the GLI1 gene to CDK4, DDIT3, STAT6, HMG2A and MDM2 genes on chromosome 12q. The lower half of the figure shows diffuse expression of CDK4 and MDM2 by immunohistochemistry in a GLI1-amplified mesenchymal tumor (400x).

Behavior

Initially thought to be an indolent neoplasm, subsequent reports have identified evidence of metastatic potential with 25% of reported cases showing metastasis. Although no definite clinico-pathologic features predictive of malignancy have been identified, increased size greater than or equal to 6 cm and mitotic activity greater than or equal to 5 per 10 high-power fields may predict increased risk of metastasis. ¹⁵

Saoud et al. ¹⁵ in their study conducted a comparative analysis of pooled survival analysis of the GLI1-fusion associated and GLI1-amplified patients and showed that GLI1-amplified cases had worse overall survival, with a remarkably shortened median progression-free survival duration.

GLI1 altered mesenchymal tumors of Head and Neck

Head and neck sites are among the most common to be involved by GLI1-altered mesenchymal tumors, comprising up to 25–30% of reported cases. ¹⁴ The study by Xu et al. ¹³ is the largest study of this entity in head and neck locations: of the 11 reported cases, the majority (8) involved the tongue. The patients ranged in age from 1 to 65 years and showed a slight male predominance. Seven of the cases showed GLI1 associated gene fusions while 4 of the cases showed GLI1 gene amplifications.

The study by Palsgrove et al. ⁸, also focusing on the head and neck, reported 5 cases of GLI1-altered mesenchymal soft tissue tumor involving tongue, base of tongue, vallecula and tonsil. Three cases harbored GLI1 associated gene fusions while 2 cases showed GLI1 gene amplifications. Of interest, this study reported strong nuclear and cytoplasmic staining for p16 in 3 of the 4 cases tested.

Overall, about 42 cases of GLI1-altered mesenchymal tumors have been reported so far in head and neck locations. ^{2, 3, 5, 6, 8, 9, 13, 14, 16, 25–28} (Table 1) Tongue appears to be the most common site of involvement among reported cases to date (26 / 42, 62%). Other sites of involvement include floor of mouth and soft tissues of the neck, along with rare cases reported in vallecula, tonsil, soft palate, orbit, hard palate, submandibular, submental and mandibular gingiva.

Table 1:

Clinico-pathologic features, genetic alterations and follow-up pf GLI1-altered mesenchymal tumors of head and neck.

Case	Study	Age	Sex	Site	Size (cm)	GLI1-alteration	FU duration (mths)	FU status
1	Dahlen et al.	27	F	tongue	0.8	ACTB::GLI1	60	NED
2	Dahlen et al.	11	M	tongue	5	ACTB::GLI1	22	NED
3	Dahlen et al.	12	F	tongue	2.4	ACTB::GLI1	120	NED
4	Xu et al.	32	F	submandibular	NA	PTCH1::GLI1	104	AWD
5	Xu et al.	38	M	tongue	NA	PTCH1::GLI1	2	NED
6	Xu et al.	37	M	submandibular	NA	ACTB::GLI1	30	NED
7	Xu et al.	1	M	tongue	NA	ACTB::GLI1	2	NED
8	Xu et al.	28	M	tongue	NA	ACTB::GLI1	NA
9	Xu et al.	14	M	tongue	NA	ACTB::GLI1	NA
10	Xu et al.	56	F	tongue	NA	MALAT1::GLI1	NA
11	Xu et al.	46	F	tongue	NA	GLI1 amplification	3	NED
12	Xu et al.	60	M	tongue	NA	GLI1 amplification	NA
13	Xu et al.	39	M	neck	N,	GLI1 amplification	36	AWD
14	Xu et al.	65	M	tongue	NA	GLI1 amplification	NA
15	Palsgrove et al.	35	M	palatine tonsil	1.5	GLI1 amplification	NA
16	Palsgrove et al.	84	M	base of tongue	NA	ACTB::GLI1	NA
17	Palsgrove et al.	53	M	oropharynx	6	ACTB::GLI1	NA
18	Palsgrove et al.	41	M	tongue	NA	ACTB::GLI1	NA
19	Palsgrove et al.	65	M	tongue	2	GLI1 amplification	NA
20	Papke Jr et al.	12	M	neck	1.5	ACTB::GLI1	NA
21	Papke Jr et al.	46	F	tongue	5.8	GLI1 amplification	17	NED
22	Papke Jr et al.	10	F	tongue	2	ACTB::GLI1	NA
23	Papke Jr et al.	1	M	tongue	0.9	NA	NA
24	Papke Jr et al.	1	M	tongue	NA	GLI1 amplification	NA
25	Yeung et al.	13	M	tongue	2.5	GLI1 amplification	19	NED
26	Yeung et al.	68	F	floor of mouth	4.1	ACTB::GLI1	81.5	DOD
27	Klubickova et al.	34	F	soft palate	3.4	PTCH1::GLI1	4	NED
28	Liu J et al.	27	M	tongue	3.5	ACTB::GLI1	16	NED
29	Liu J et al.	4	M	tongue	3	GLI1 amplification	11	NED
30	Liu J et al.	17	M	tongue	1	GLI1 amplification	5	NED
31	Liu J et al.	8	M	floor of mouth	2	ACTB::GLI1	40	AWD
32	Liu J et al.	34	M	floor of mouth	NA	MALAT1::GLI1	NA
33	Liu Y et al.	57	F	gingiva	4	GLI1 amplification	NA
34	Na et al.	32	M	tongue	2.8	GLI1 amplification	NA
35	Godse et al.	54	F	orbit	NA	GLI1 amplification	6	NED
36	Dehner et al.		M	tongue		ACTB::GLI1	NA
37	Zhong et al.	56	M	tongue	6	GLI1 rearrangement	68	AWD
38	Kerr et al.	2	M	tongue	NA	ACTB::GLI1	NA
39	Kerr et al.	62	M	hard palate	NA	ACTB::GLI1	NA
40	Kerr et al.	37	F	floor of mouth	NA	PTCH1::GLI1	NA
41	Kerr et al.	51	F	neck	0.9	ACTB::GLI1	12	NED
42	Shahabi et al.	82	F	submental	5.3	PTCH1::GLI1	4	NED

F, female; M, male; NA, not available; NED, no evidence of disease; AWD, alive with disease; DOD, died of disease.

Tumors showed a predilection to occur in males (M:F 2:1, 28/42). Tumors occurred over a wide age range of 1–82 years with a median age of 35 years.

Molecular alterations

Most of the cases (26/42, 62%) had a GLI1 associated gene fusion, while the rest of the cases (16/42, 38%) showed GLI1 gene amplifications. Of the GLI1 gene fusions noted, 19 of 26 (73%) were ACTB::GLI1, 5 (19%) were PTCH1::GLI1 and 2(8%) were MALAT1::GLI1 gene fusion.

Follow-up

Follow-up data has been reported in 21 cases ranging from 2 to 120 months with a median follow-up of 17 months. 16 of the 21 patients (76%) had no evidence of disease at the time of last follow-up, 4 patients (19%) were Alive with Disease (AWD), and 1 patient (5%) had succumbed to the disease (DOD). Yeung et al., ¹⁴ in their meta-analysis of reported cases, identified 22% of cases of the head and neck showing metastasis.

Diagnostic Paradigm

A diagnostic paradigm aids in the identification of this entity. (Figure 4) If the lesion shows the characteristic morphology of small to medium sized cells in a nested pattern with intervening rich vascularity, immunohistochemical stains for GLI1, CDK4, MDM2 and STAT6 should be included in the immunohistochemical panel along with the rest of the stains. If the tumor cells are positive for GLI1, CDK4, MDM2 and/or STAT6, additional cytogenetic (FISH) or DNA sequencing studies to detect amplification of GLI1, DDIT3, CDK4 and MDM2 genes may help in the diagnosis of GLI1-amplified mesenchymal tumor. If GLI1 immunohistochemical stain is positive, while other stains for CDK4, MDM2 and/or STAT6 are negative, additional RNA sequencing to detect possible GLI1-gene fusion could be pursued.

Figure 4:

Diagnostic paradigm of GLI1-altered mesenchymal tumor, where a characteristic morphologic pattern leads to subsequent immunohistochemical, cytogenetic and molecular work-up that may aid in the diagnosis of this entity.

Summary

GLI1-altered mesenchymal tumors are a rare group of recently described tumors which tend to occur in various locations including head and neck, soft tissues and within viscera. Molecularly, they are characterized by either GLI1-associated gene fusions or by GLI1-gene amplifications. Regardless of the site or the molecular alteration, the tumors show distinctive morphologic features including uniform round to ovoid cells in a nested growth pattern, admixed with a rich capillary vascular network. The immunoprofile of these tumors is rather nonspecific. Although the morphology provides a clue, cytogenetic and molecular fusion analysis are the main ancillary studies that confirm the diagnosis. Although no definite morphologic features predict the behavior of this tumor, recent studies suggest that GLI1-amplified tumors may behave worse than tumors with GLI1-gene fusions.