Classification of follicular-patterned thyroid lesions using a minimal set of epigenetic biomarkers.

Objective: The minimally invasive fine-needle aspiration cytology (FNAC) is the current gold standard for the diagnosis of thyroid nodule malignancy. However, the correct discrimination of follicular neoplasia often requires more invasive diagnostic techniques. The lack of suitable immunohistochemical markers to distinguish between follicular thyroid carcinoma and other types of follicular-derived lesions complicates diagnosis, and despite most of these tumours being surgically resected, only a small number will test positive for malignancy. As such, the development of new orthogonal diagnostic approaches may improve the accuracy of diagnosing thyroid nodules. Design: This study includes a retrospective, multi-centre training cohort including 54 fresh-frozen follicular-patterned thyroid samples and two independent, multi-centre validation cohorts of 103 snap-frozen biopsies and 33 FNAC samples, respectively. Methods: We performed a genome-wide genetic and epigenetic profiling of 54 fresh-frozen follicular-patterned thyroid samples using exome sequencing and the Illumina Human DNA Methylation EPIC platform. An extensive validation was performed using the bisulfite pyrosequencing technique. Results: Using a random forest approach, we developed a three-CpG marker-based diagnostic model that was subsequently validated using bisulfite pyrosequencing experiments. According to the validation cohort, this cost-effective method discriminates between benign and malignant nodules with a sensitivity and specificity of 97 and 88%, respectively (positive predictive value (PPV): 0.85, negative predictive value (NPV): 0.98). Conclusions: Our classification system based on a minimal set of epigenetic biomarkers can complement the potential of the diagnostic techniques currently available and would prioritize a considerable number of surgical interventions that are often performed due to uncertain cytology. Significance statement: In recent years, there has been a significant increase in the number of people diagnosed with thyroid nodules. The current challenge is their etiological diagnosis to discount malignancy without resorting to thyroidectomy. The method proposed here, based on DNA pyrosequencing assays, has high sensitivity (0.97) and specificity (0.88) for the identification of malignant thyroid nodules. This simple and cost-effective approach can complement expert pathologist evaluation to prioritize the classification of difficult-to-diagnose follicular-patterned thyroid lesions and track tumor evolution, including real-time monitoring of treatment efficacy, thereby stimulating adherence to health promotion programs.

Usefulness of urinary biomarkers to estimate the interstitial fibrosis surface in diabetic nephropathy with normal kidney function.

BACKGROUND: Kidney biopsies of patients with diabetic nephropathy (DN) and normal kidney function may exhibit interstitial fibrosis (IF) without reduction of glomerular filtration rate (GFR) because of hyperfiltration. The aim of our study was to analyse the performance of a set of biomarkers of tubular injury to estimate the extent of IF in patients with DN and normal kidney function. METHODS: This cross-sectional study included 118 adults with DN diagnosed by kidney biopsy and GFR ≥90 mL/min/1.73 m2 and a control group of healthy subjects. We measured the urinary excretion of monocyte chemoattractant protein-1 (MCP-1) neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver-type fatty acid-binding protein (L-FABP), ß2-microglobulin and dickkopf-3 protein (DKK-3) at the time of kidney biopsy. GFR was measured by chromium-51 labeled ethylenediamine tetraacetic acid (Cr-EDTA) (measured GFR). IF was quantified using a quantitative morphometric procedure. Predictive multivariate models were developed to estimate the IF surface. RESULTS: Patients with DN showed significantly higher levels of DKK-3, MCP-1 and L-FABP and significantly lower levels of epidermal growth factor (EGF) than healthy controls. There were no significant between-group differences in the levels of ß2-microglobulin, KIM-1 or NGAL. IF was negatively associated with EGF and positively with age, proteinuria, MCP-1, DKK-3 and L-FABP, but not with ß2-microglobulin, KIM-1, NGAL or GFR. The best model to predict IF surface accounted for 59% of its variability and included age, proteinuria, EGF, DKK-3 and MCP-1. CONCLUSIONS: Our study provides a model to estimate the IF in DN that can be useful to assess the progression of IF in patients with normal kidney function.

CD44-negative parietal-epithelial cell staining in minimal change disease: association with clinical features, response to corticosteroids and kidney outcome.

BACKGROUND: Activation of parietal-epithelial cells (PECs) with neo-expression of CD44 has been found to play a relevant role in the development of focal and segmental glomerulosclerosis (FSGS). The aim of this study was to analyse whether the expression of CD44 by PECs in biopsies of minimal change disease (MCD) is associated with the response to corticosteroids, with kidney outcomes and/or can be considered an early sign of FSGS. METHODS: This multicentric, retrospective study included paediatric and adult patients with MCD. Demographic, clinical and biochemical data were recorded, and biopsies were stained with anti-CD44 antibodies. The association between PECs, CD44 expression and the response to corticosteroids, and kidney outcomes were analysed using logistic, Kaplan-Meier and Cox regression analyses. RESULTS: A total of 54 patients were included: 35 (65%) <18 years and 19 (35%) adults. Mean follow-up was 68.3 ± 37.9 months. A total of 19/54 patients (35.2%) showed CD44-positive staining. CD44-positive patients were younger (14.5 ± 5 versus 21.5 ± 13, P = 0.006), and showed a higher incidence of steroid-resistance [11/19 (57.8%) versus 7/35 (20%), P = 0.021; odds ratio: 5.5 (95% confidence interval 1.6-18), P = 0.007] and chronic kidney disease [9/19 (47.3%) versus 6/35 (17.1%), P = 0.021; relative risk: 3.01 (95% confidence interval 1.07-8.5), P = 0.037]. Follow-up re-biopsies of native kidneys (n = 18), identified FSGS lesions in 10/12 (83.3%) of first-biopsy CD44-positive patients versus 1/6 (16.7%) of first-biopsy CD44-negative patients (P = 0.026). CONCLUSIONS: In patients with a light microscopy pattern of MCD, CD44-positive staining of PECs is associated with a higher prevalence of steroid resistance and worse kidney outcomes, and can be considered an early sign of FSGS.