Diagnostic tools in the differential diagnosis of giant cell-rich lesions of bone at biopsy

Abstract

The diagnosis of giant cell-rich lesions of bone can be challenging if radiological findings are ambiguous and tissue of the biologically deciding component is underrepresented in biopsy specimens. The frequent association of giant cell tumor of bone (GCT) and chondroblastoma (CB) with (secondary) aneurysmal bone cysts (ABC) may further impede correct classification. The present study evaluates the potentials and limitations of mutation-specific histone H3.3 and DOG1 immunohistochemistry, Sanger-/next generation sequencing (NGS) and FISH analysis in the differential diagnosis of 23 GCT, 14 CB and 19 ABC. All morphologically typical GCT and CB harbored mutations in the H3F3A or H3F3B gene, respectively. These were, except for one uncommon G34L mutation in a GCT, reliably and specifically detected by mutation-specific H3.3 G34W or H3.3 K36M immunohistochemistry and DNA sequencing. In the diagnostic substantiation of CB, DOG1 staining was less sensitive compared to H3.3 K36M immunohistochemistry. 47% of ABC specifically showed translocations of the USP6 gene, while mutations in H3F3A/B were absent. Based on the results of this study, we conclude that mutation-specific H3.3 immunohistochemistry (selectively complemented with NGS-based DNA sequencing) and USP6 FISH analysis enable a reliable diagnostic distinction of GCT, CB and ABC of morphologically and radiologically difficult cases.

Keywords: H3F3A/B; aneurysmal bone cyst; chondroblastoma; diagnostic algorithm; giant cell tumor of bone.

Figure 1. Clustered overview of clinical and pathological patient data, the results of the immunostainings (H3.3 G34W, H3.3 K36M, DOG1), the mutational H3F3A/B status as detected by DNA sequencing and the result of the USP6 break-apart FISH

Cases are categorized in diagnosis groups (GCT, CB, ABC).

Figure 2

(A) Representative staining results and DNA sequence electropherograms. Top: GCT with G34W alteration (case #3). Bottom: CB with K36M alteration (case #25). Corresponding DNA sequence electropherograms are shown below. (B, C) Immunohistochemical staining and corresponding Sanger and NGS sequencing results in two diagnostically challenging cases of GCT. (B) GCT with a G34L alteration (case #15). (C) Top: GCT with low mutation frequency (7.5%) demonstrated by immunohistochemistry and NGS, not detectable by Sanger sequencing. Bottom: X-ray and MRI image (case #21).

Figure 3

(A) Immunohistochemical staining and USP6 FISH results shown for primary ABC (case #39). (B) Left: Immunohistochemical H3.3 K36M staining results in a case with extensive secondary ABC and underrepresented lesional CB tissue in the biopsy material (case #27). No H3F3B mutation could be detected by DNA sequencing due to the low fraction of mutated lesional cells. Right side: X-ray and MRI image.

Figure 4. Proposed diagnostic algorithm for radiologically and morphologically ambiguous giant cell-rich lesions of bone at biopsy: Immunohistochemistry with mutation-specific H3.3 antibodies is recommended as a starting point for the detection of underlying GCT or CB

In case of negativity, USP6 FISH is suggested to confirm/exclude primary ABC. DNA sequencing of the H3F3A and H3F3B genes is required only in immunohistochemically H3.3 G34W- and K36M-negative and non USP6-rearranged cases to rule out secondary ABC with underlying GCT or CB with a non-typical H3F3A and/or H3F3B gene alteration.