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. 2024 Jan 15;16(1):304-313.
doi: 10.62347/QOBT7285. eCollection 2024.

Hypermethylated SHOX2 in circulating cell-free DNA post renal cell carcinoma surgery as TNM-independent biomarker for recurrence risk

Thomas Büttner  1 , Romina Zarbl  2 , Philipp Krausewitz  1 , Sebastian Strieth  2 , Glen Kristiansen  3 , Markus Eckstein  4 , Damian J Ralser  5   6 , Michael Hölzel  6 , Manuel Ritter  1 , Jörg Ellinger  1 , Dimo Dietrich  2 , Niklas Klümper  1   6
Affiliations

Hypermethylated SHOX2 in circulating cell-free DNA post renal cell carcinoma surgery as TNM-independent biomarker for recurrence risk

Thomas Büttner et al. Am J Transl Res. .

Abstract

Introduction: Adjuvant immune checkpoint inhibitor trials in renal cell carcinoma (RCC) call for improved recurrence risk stratification. Due to limitations of circulating tumor DNA (ctDNA) use in RCC, the use of hypermethylated SHOX2 gene (mSHOX2) in circulating cell-free DNA is explored as a surrogate marker for identifying high-risk patients after RCC surgery.

Methods: Liquid biopsies were collected post-surgery from 45 RCC patients (mean duration 4.3 days). Real-time polymerase chain reaction was used to analyze SHOX2 methylation in circulating cell-free DNA. Patients were categorized as mSHOX2 positive or negative by cut-off. Metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival (OS) were assessed using Cox regression and Log-rank analyses (median follow-up time: 60 months).

Results: 17 patients were mSHOX2 positive, showing unfavorable OS/CSS (Log-rank P = 0.004 and 0.02) and nearly 6-fold higher recurrence risk (hazard ratio 5.89, 95% CI 1.46-23.8). Multivariable Cox analysis confirmed mSHOX2 as an independent recurrence risk factor, disregarding TNM-based stratification.

Conclusions: mSHOX2 effectively identifies high-risk RCC patients post-surgery, indicating minimal residual disease. This easy to implement biomarker has potential for guiding of adjuvant therapy decisions.

Keywords: Renal cell carcinoma; SHOX2; adjuvant therapy; biomarker; recurrence.

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Conflict of interest statement

DD has been an employee of Epigenomics AG, Berlin, Germany. This company aims to commercialize DNA methylation biomarker SHOX2. DD is co-inventor and owns patents on methylation biomarkers and related technologies. DD receives inventor’s compensation from Epigenomics AG.

Figures

Figure 1
Figure 1
Kaplan-Meier estimators of survival after renal cell carcinoma surgery: All 45 patients were divided by methylation status of short stature homeobox 2 (SHOX2) gene in circulating cell-free DNA into either hypermethylated (mSHOX2-positive) or non-hypermethylated (mSHOX2-negative). Significant survival benefit was shown for mSHOX2-negative patients in: (A) Overall survival (Log rank P = 0.004), (B) Cancer-specific-survival (Log rank P = 0.017) and (C) Metastasis-free-survival (MFS, Log rank P = 0.005). For MFS analysis primary metastatic cases were excluded. (D) By composing mSHOX2 with high-risk criteria according to KEYNOTE-564 trial (pT2, G4; pT3-4; pN+, pM+ [19]) one event of recurrence is missed out (Log rank P < 0.001).
Figure 2
Figure 2
Kaplan-Meier estimators of survival after renal cell carcinoma surgery. After exclusion of primary metastatic cases, 39 patients are divided by methylation status of short stature homeobox 2 (SHOX2) gene in circulating cell-free DNA into either hypermethylated (mSHOX2-positive) or non-hypermethylated (mSHOX2-negative). A significant survival benefit was shown for mSHOX2-negative patients in (A) overall survival (Log rank P = 0.025) with a non-significant trend for (B) cancer-specific-survival (Log rank P = 0.11).
Figure 3
Figure 3
Kaplan-Meier estimator of metastasis-free survival in the low-risk subgroup after renal cell carcinoma surgery. Additional analysis including only patients with low risk of recurrence according to KEYNOTE-564 trial (pT1-2, G1-3, N0, M0) [19]. Hypermethylation of short stature homeobox 2 (mSHOX2) in circulating cell-free DNA was associated with impaired MFS (Log rank P < 0.01).
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