Analysis of Pleckstrin Homology Domain Containing S1 (PLEKHS1) Promoter Mutations in Pre-Operative Thyroid Nodule Samples

Introduction

• Pleckstrin homology domain containing S1 (PLEKHS1) (Figure 1) is a poorly characterized protein coding gene, whose C593T and G590A promoter mutations are associated with increased risk of lymph node and distant metastases,¹ RAI refractoriness, and shorter survival¹ in differentiated thyroid cancer independent of TERT promoter (TERTP) mutations.
• The diagnostic and prognostic significance of these mutations in thyroid nodules is unknown.
• Here we assess the potential impact of pre-operative detection of PLEKHS1 promoter mutations in thyroid nodules.

Methods

• Targeted C593T and G590A PLEKHS1 promoter mutations were assessed in 9,279 patient samples from April 2023 to June 2024 in indeterminate thyroid nodules (ITNs) with Bethesda (B) III/IV cytology (ITN) and Afirma GSC suspicious (GSC-S) results or with B V/VI cytology.
• A subset of consecutive cases positive for the targeted PLEKHS1 promoter mutations with surgical histology (n=20) were analyzed for co-occurring molecular alterations and pathology outcomes.
• Pathology outcomes were collected under WCG IRB #1384712.

Results

PLEKHS1 promoter mutations were assessed in 9,279 patient samples.

The demographics of the 9,279 samples tested for the PLEKHS1 C593T and G590A hotspot promoter mutations, along with the proportion with TERT promoter (TERTP) or BRAF p.V600E mutations are shown in Table 1.

PLEKHS1 promoter mutations were positive in 60/9,279 (0.6%) of patient samples. The proportions of each hotspot mutation (C593T and G590A) assessed, and concomitant positive TERTp mutations and BRAF p.V600E mutations are shown in Table 2.

The proportion of PLEKHS1 positive cases was highest in samples with Bethesda VI cytology (Table 3).

Table 4 shows the case findings for 20 nodules with PLEKHS1 positive mutations (15 from GSC-S ITN and 5 from BV/VI nodules). 40% (6/15) of ITN were malignant and met American Thyroid Association (ATA) criteria for low-risk cancer; 5/6 (83%) had co-alterations: KRAS p.G12D, DICER1 p.E1705K, NRAS p.Q61R + PIK3CA p.E545K + TERT C228T, NRAS p.Q61R, and FGFR2::VCL fusion. 2/9 (22%) benign ITN had co-alterations (PAX8::GLIS3 fusion and NRAS p.Q61R). Only 1/8 (12.5%) ITN with an isolated PLEKHS1 mutation was malignant (minimally invasive oncocytic carcinoma). All 5 BV/VI nodules were malignant and met ATA criteria for high (2), intermediate (1), or low (2) risk cancer.

Conclusion

• PLEKHS1 promoter mutations are rare in thyroid nodules undergoing molecular testing.
• In contrast with data in metastatic PTC, isolated PLEKHS1 mutations detected in ITNs do not predict higher risk of cancer nor aggressive histology as compared with other Afirma GSC-S nodules.
• Further studies are needed to clarify the role of PLEKHS1 promoter mutations in thyroid nodules and differentiated thyroid cancer to harness the full potential of molecular information for improving patient care.