# Development of a multi-analyte diagnostic assay to improve the risk stratification of indeterminate thyroid nodules

> **NIH NIH R44** · ALAUNUS BIOSCIENCES, INC. · 2023 · $696,026

## Abstract

Abstract
Widespread use of sensitive imaging in clinical practice has resulted in incidental thyroid nodules
being discovered with increasing frequency, creating a unique opportunity to detect and remove
lesions at early stages of malignancy. The majority of these incidental nodules remain benign and
asymptomatic, but 10-20% are found to be malignant on surgical excision, resulting in a critical need
for improved diagnostics, as 75% of patients currently undergo unnecessary thyroidectomies to
remove benign nodules. The current standard of care includes collecting a tissue biopsy by
ultrasound-guided fine needle aspiration: these samples are tested by cytology, which looks for
aberrant cells, and for genetic risk markers. These diagnostic tests are characterized by poor
specificity, and a third of all tested nodules are still classified as indeterminate and need to be
resected for a pathological diagnosis. We originally hypothesized that dysregulated proteolysis, a
type of enzyme activity that is a hallmark of invasive cancer, might yield discriminating levels of
activity in FNA tissue from benign and malignant nodules. In Phase I work, we leveraged the Alaunus
Biosciences, Inc. diagnostic pipeline, a platform that allows innovative proteomics-based biomarker
discovery, to identify and characterize a set of protease activities (functional markers) that are
significantly increased in malignant nodules. We then developed fluorogenic substrate assays to
monitor these activities using small volumes of biological samples. In parallel, we also discovered
additional mass-based markers that can be measured with standard antibody-based assays. Our
investigational multi-analyte diagnostic assay has the potential to differentiate malignant from benign
thyroid tissue with sensitivity and specificity >90%. In this Phase II proposal, we aim to perform
technical and clinical validation studies required to advance the assay to readiness for clinical use in
the early diagnosis of indeterminate thyroid nodules. In Aim 1, we will benchmark the clinical utility of
our selected protein and functional biomarkers in surgical tissue to evaluate scoring, establish
diagnostic thresholds, and report on key assay parameters. In Aim 2, we will perform a
comprehensive technical assessment of the laboratory performance of the assay to satisfy industry
standards. In Aim 3, we will create an independent cohort of FNA tissue samples, through our IRB-
approved biobanking efforts at UCSF, UCLA and IU, to be used for a robust prospective validation of
the clinical usefulness of the assay. Our goal is to develop a rapid assay that improves patient
stratification over current standard diagnostic markers, guides clinical decision-making to avoid
unnecessary surgical intervention, and transforms the clinical management of these challenging
lesions.

## Key facts

- **NIH application ID:** 10684875
- **Project number:** 5R44CA254649-03
- **Recipient organization:** ALAUNUS BIOSCIENCES, INC.
- **Principal Investigator:** GISELLE M KNUDSEN
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $696,026
- **Award type:** 5
- **Project period:** 2022-08-15 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10684875

## Citation

> US National Institutes of Health, RePORTER application 10684875, Development of a multi-analyte diagnostic assay to improve the risk stratification of indeterminate thyroid nodules (5R44CA254649-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10684875. Licensed CC0.

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