A clinical scoring system for selection of patients for PTEN mutation testing is proposed on the basis of a prospective study of 3042 probands

Abstract

Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome are allelic, defined by germline PTEN mutations, and collectively referred to as PTEN hamartoma tumor syndrome. To date, there are no existing criteria based on large prospective patient cohorts to select patients for PTEN mutation testing. To address these issues, we conducted a multicenter prospective study in which 3042 probands satisfying relaxed CS clinical criteria were accrued. PTEN mutation scanning, including promoter and large deletion analysis, was performed for all subjects. Pathogenic mutations were identified in 290 individuals (9.5%). To evaluate clinical phenotype and PTEN genotype against protein expression, we performed immunoblotting (PTEN, P-AKT1, P-MAPK1/2) for a patient subset (n = 423). In order to obtain an individualized estimation of pretest probability of germline PTEN mutation, we developed an optimized clinical practice model to identify adult and pediatric patients. For adults, a semiquantitative score-the Cleveland Clinic (CC) score-resulted in a well-calibrated estimation of pretest probability of PTEN status. Overall, decreased PTEN protein expression correlated with PTEN mutation status; decreasing PTEN protein expression correlated with increasing CC score (p < 0.001), but not with the National Comprehensive Cancer Network (NCCN) criteria (p = 0.11). For pediatric patients, we identified highly sensitive criteria to guide PTEN mutation testing, with phenotypic features distinct from the adult setting. Our model improved sensitivity and positive predictive value for germline PTEN mutation relative to the NCCN 2010 criteria in both cohorts. We present the first evidence-based clinical practice model to select patients for genetics referral and PTEN mutation testing, further supported biologically by protein correlation.

Figure 1

Consolidated PTEN Mutation Spectrum Distribution and number of substitutions (missense and/or nonsense), small insertion mutations, and small deletion mutations across the gene. In the top panel, blue bars represent missense mutations and red bars represent nonsense mutations. In the second panel, the blue arrowheads represent small deletions and the red arrowheads represent small insertions along the gene. Complex mutations (indels, splice-site mutations, and large deletions) and promoter mutations are not depicted. For both panels, frequencies of both the substitution mutations and the insertion/deletion mutations are shown on the left. The bar below corresponds to the multiple exons of the PTEN cDNA molecule, with exon 1 on the left to exon 9 on the right, allowing for matching of mutation to exon. As evident, exons 5, 7, and 8 are sites of common mutations.

Figure 2

CC Score Nomogram for Obtaining a Corresponding Individual Point Pretest Probability of Germline PTEN Mutation The CC score is first derived by a sum of the weights of positive features that is provided in Table 4. To illustrate, three hypothetical cases are presented, each corresponding to a CC score of 5 points, 10 points, and 15 points, respectively. Case 1 may present with breast cancer at age 55 (1 point), with background of thyroid cancer at age 44 (4 points), with a final score of 5 and corresponding point probability < 1%. Case 2 may present with breast cancer at age 38 (4 points) and concurrent macrocephaly (6 points), with a final score of 10 and corresponding point probability of 3%. Case 3 may present with a single hamartomatous gastrointestinal polyp (10 points) found on endoscopy, Hashimoto's thyroiditis (4 points), and lipomas (1 point), for a final score of 15 and corresponding point probability of 10%.

Figure 3

A Calibration Plot for Model-Based Predicted Probabilities of PTEN Mutation and Actual Outcomes The calibration plot shows excellent bias-corrected correlation between observed and predicted values for the developed model within the CC data set, suggesting good internal calibration for the CC score model, that is, individual predicted and actual outcomes are similar. The dashed line at 45° (y = x) represents ideal agreement between observed and predicted probabilities of PTEN mutation.

Figure 4

A Graph Showing that the CC Score Yields Superior Predictions Relative to the NCCN Criteria in Both the CC and the OSU Data Sets Each data set shows corresponding likelihood ratio chi-squares for the NCCN criteria, the CC score, and a full model comprised of both criteria. The CC score confers statistically significant benefit to the NCCN criteria in PTEN mutation prediction, but the NCCN criteria do not confer benefit to the CC score. Higher adequacy indices are observed for the CC score relative to the NCCN criteria in both data sets.

Figure 5

Bar Plot Showing Expression by Immunoblot of Downstream Readout Molecules of PTEN Function for Immortalized Lymphoblasts from 423 Patients A statistically significant decrease in PTEN protein expression and increase in phospho-AKT1 expression are noted for samples with pathogenic mutations. Standard errors are shown in the error bar. P values are derived from ANOVA testing.

Figure 6

The Relationship between the CC Score and PTEN Protein Quantitation This relationship between CC score and PTEN protein levels is classified by quintiles, lowest to highest from left to right of each graph, with an inverse association that is demonstrated between rising PTEN protein expression and decreasing mean CC score in the full data set on linear regression (p < 0.001). Standard errors are shown by error bar.

Figure 7

The Relationship between the CC Score and PTEN Protein Quantitation after Exclusion of Patients with PTEN Mutations The relationship between CC score and PTEN expression levels is classified by quintiles, lowest to highest from left to right of each graph. An inverse association between rising PTEN protein expression and decreasing CC score is seen in samples derived from patients without pathogenic germline PTEN mutations (p = 0.036). Standard errors are shown by error bar.