GLI1-amplifications expand the spectrum of soft tissue neoplasms defined by GLI1 gene fusions

Abstract

GLI1 fusions involving ACTB, MALAT1, and PTCH1 genes have been recently reported in a subset of malignant soft tissue tumors with characteristic monomorphic nested epithelioid morphology and frequent S100 positivity. However, we encountered a group of morphologically similar soft tissue tumors lacking the canonical GLI1 gene fusions and sought to investigate their genetic abnormalities. A combined approach including RNA sequencing, targeted exome sequencing and FISH methodologies were used to identify potential novel genetic abnormalities. Ten patients (five females, five males) with an age range of 4-65 years (median 32.5) were identified. Tumors were located in the soft tissues of the limbs, trunk and head and neck, with one each in the tongue and lung. Histologically, tumors revealed ovoid to epithelioid cells arranged in a distinctive nested-trabecular pattern, separated by thin septa and a delicate vascular network. Two cases showed areas of increased nuclear pleomorphism and focal fascicular spindle cell growth. Four tumors showed a high mitotic count (≥15/10 HPFs), with necrosis seen in three of them. Lymphovascular invasion was noted in two cases. No consistent immunoprofile was detected, with positivity for CD56 (six cases), S100 (four cases), SMA (two cases), and pan-CK (one case). FISH showed GLI1 (12q13.3) gene amplification in all 10 cases, with co-amplification of CDK4 (12q14.1) in nine (90%) and MDM2 (12q15) in eight (80%) cases. Targeted exome sequencing performed in three cases confirmed the GLI1, CDK4, and MDM2 co-amplification. Only one case showed the presence of both GLI1 break-apart and amplification, although no gene partner was detected. Our findings suggest that GLI1 amplification, often associated with co-amplifications of CDK4 and MDM2 genes, may represent an alternative genetic mechanism of GLI1 oncogenic activation akin to GLI1 fusions, defining the pathogenesis of an emerging group of malignant soft tissue tumors with a distinctive nested growth pattern and variable immunoprofile.

Figure 1:. Pathologic and FISH findings of GLI1 amplified tumors.

A well-circumscribed lesion with distinct lobulated architecture (A) showing monomorphic ovoid cells arranged in a cords separated by delicate capillaries (B) (Case 1, 4/F, shoulder). C-I: Images from Case 2 (10/M, finger) of a superficial dermal nodule with a nodular growth pattern (C), in areas protruding into dilated vascular spaces (D) and showing tumor cells with clear cytoplasm in a nested arrangement (E). Immunohistochemical stains for CDK4 (F) and MDM2 (G) were strongly positive. FISH studies showing (H) amplification and break-apart of GLI1 gene (green- 5’-centromeric separated from red-3’-telomeric, arrow; the latter red signal being also being amplified) and (I) Co-amplifications of CDK4 and MDM2 genes (orange-MDM2, red-CDK4, arrow).

Figure 2:. Morphologic and immunohistochemical spectrum of GLI1 amplified tumors.

(A-C) Case 6 (39/M, neck) showed a biphasic histology with the epithelioid nested growth (A) in abrupt transition with the bland spindle cell component arranged in short fascicles (B); immunohistochemical stain for SMA (C) showing diffuse positivity. (D-F) Case 7 (51/F, back) showing (D) bland epithelioid cells arranged in single files in a collagenous stroma. Other areas showed more solid growth with increased atypia and mitotic activity (E). Immunohistochemical stain for S100 (F) showing patchy strong positivity. (G-I) Images from Case 8 (54/F, elbow) showing distinctive multinodular growth (G), which on high power reveals a tight nested growth of uniform epithelioid cells (H). Immunohistochemical stain for CDK4 (I) showing strong and diffuse positivity.

Figure 3:. Morphologic and immunohistochemical spectrum of GLI1 amplified tumors.

Case 4 (23/F, thigh) showing tumor lobules with cribriform or distinctive rosette-like pattern, separated by a fibrotic stroma (A), with high power also showing cord-like and trabecular pattern (B); (C-D) Case 5 (26/F, lung) showing tumor cells in a trabecular pattern with intervening sinusoidal pattern. Immunohistochemical stain for CD56 (D) showing positivity. (E-I) Case 10 (65/M, tongue) showed a compact growth, with vague nested growth of epithelioid cells separated by fibrotic stroma; tumor cells were uniform and showed clear cytoplasm (E). Diffuse positivity for MDM2 (F), CDK4 (G) and STAT6 (H) immunohistochemical stains was noted. FISH showed GLI1 gene (green-centromeric, red-telomeric) amplification (arrows)(I).

Figure 4.. GLI1 mRNA upregulation in tumors with GLI1 genetic abnormalities.

A high GLI1 mRNA overexpression was present in cases with both GLI1 amplifications (cases’ 3 and 8) and GLI1 fusions (3 cases).(7) One dedifferentiated liposarcoma with GLI1/CDK4/MDM2 co-amplification showed a lower level of upregulation. In contrast 2 dedifferentiated liposarcomas with MDM2/CDK4 amplification and other sarcoma types showed no increased GLI1 mRNA levels. Higher levels of STAT6 and HMGA2 expression were noted in cases with GLI1 amplification suggesting their co-amplification gene status; in contrast to tumors with GLI1 fusions. The genomic positions and relationship of the genes located on the 12q13–15 locus (lower panel).