SBIR-STTR Award

A dramatic increase in the number of patients being diagnosed with incidental thyroid nodules through different diagnostic imaging modalities has created a unique opportunity to detect and remove lesions at early stages of malignancy. This also creates a critical need for improved diagnostics, as 80% of patients currently undergo unnecessary thyroidectomies because their nodules were benign. 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 perform poorly, consequently a third of all nodules are still classified as indeterminate and are removed for a pathological diagnosis. We 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. The Alaunus Biosciences, Inc. diagnostic pipeline takes advantage of a substrate profiling technology developed in the Craik Laboratory at UCSF, a platform that allows innovative proteomics-based biomarker discovery. Using this technology, we have previously identified and characterized a proprietary set of protease activities that are significantly increased in pre-malignant pancreatic cysts; we then developed fluorogenic substrate assays to monitor these activities using small volumes of liquid biopsy (cyst fluid). In this Phase I proposal we aim to apply our discovery pipeline to a retrospective cohort of pathologically-confirmed thyroid cancer and matched normal tissue, to identify protease activities that are specific to malignant tissue. In Aim 1, we will profile thyroid tumor-specific proteolytic signatures, and identify candidate enzyme markers responsible for the observed proteolytic activity. In Aim 2, we will design and rationally optimize specific fluorogenic substrates for these proteases that can be used to rapidly detect malignant nodules in FNA samples. Lead candidate substrates will proceed to a robust validation of their clinical utility as diagnostic tools, by benchmarking a prototype assay against the current clinical standard guidelines. Our goal is to develop a rapid assay that improves patient stratification over current standard diagnostic markers and guides clinical decision-making to avoid unnecessary surgical intervention. If successful, this proposal will lay the groundwork for an actionable diagnostic test that will enable improved risk stratification of indeterminate thyroid nodules, and transform the clinical management of these challenging lesions.

Public Health Relevance Statement:
Project Narrative Early diagnosis is the single most important tool to delay or avoid cancer-related mortality. Many people undergoing diagnostic imaging today are found to have nodules in their thyroid, which in some cases are malignant; however, it is currently very difficult to predict which nodules are true malignant lesions and which will remain benign and asymptomatic. Using our previously validated technology, we aim to discover biomarkers to differentiate benign from malignant thyroid nodules, to facilitate early detection of malignancy, as well as to avoid unnecessary and costly surgeries for cases with benign nodules.

Project Terms:
Biological Assay; Assay; Bioassay; Biologic Assays; Biological Sciences; Biologic Sciences; Bioscience; Life Sciences; Biopsy; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Malignant neoplasm of thyroid; Malignant Thyroid Gland Neoplasm; Malignant Tumor of the Thyroid; Malignant Tumor of the Thyroid Gland; Thyroid Cancer; Cells; Cell Body; Classification; Systematics; Cytology; Diagnosis; Diagnostic Imaging; Engineering; Enzymes; Enzyme Gene; Fluorescence; Fluorogenic Substrate; Foundations; Future; Goals; Laboratories; Lead; Pb element; heavy metal Pb; heavy metal lead; Libraries; mortality; Nodule; Pancreatic Cyst; Pancreas Cyst; Patients; Peptide Hydrolases; Esteroproteases; Peptidases; Protease Gene; Proteases; Proteinases; Proteolytic Enzymes; Sensitivity and Specificity; Mass Spectrum Analysis; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Technology; Testing; Thyroid Gland; Thyroid; Thyroid Head and Neck; thyroid neoplasm; Thyroid Gland Neoplasm; Thyroid Gland Tumor; Thyroid Tumor; Thyroid Nodule; Thyroid Gland Nodule; Thyroidectomy; Tissues; Body Tissues; Ultrasonography; Echography; Echotomography; Medical Ultrasound; Ultrasonic Imaging; Ultrasonogram; Ultrasound Diagnosis; Ultrasound Medical Imaging; Ultrasound Test; diagnostic ultrasound; sonogram; sonography; sound measurement; ultrasound; ultrasound imaging; ultrasound scanning; United States; Generations; Diagnostic tests; Guidelines; base; improved; Benign; Clinical; Malignant; Malignant - descriptor; Phase; Biochemical; prognostic; Lesion; enzyme activity; Normal Tissue; Normal tissue morphology; tool; Diagnostic; Peptide Library; Scanning; Clinic; Best Practice Analysis; Benchmarking; Operative Procedures; Surgical; Surgical Interventions; Surgical Procedure; surgery; Operative Surgical Procedures; early detection; Early Diagnosis; experience; Performance; Protein Cleavage; Proteolysis; FRET; Förster Resonance Energy Transfer; Fluorescence Resonance Energy Transfer; cohort; Cyst Fluid; Biopsy Sample; Biopsy Specimen; Abscission; Extirpation; Removal; Surgical Removal; resection; Excision; Sampling; Proteomics; image-based method; imaging method; imaging modality; Tissue Sample; FNA; Fine Needle Aspirate; Fine-Needle Aspiration; Fine needle aspiration biopsy; Detection; Molecular Analysis; precancerous; premalignant; Stratification; Cancer Patient; Clinical Management; Genetic Risk; Risk Marker; SBIR; Small Business Innovation Research; Small Business Innovation Research Grant; Validation; Pathologic; Monitor; Resected; Active Follow-up; active followup; follow up; followed up; followup; follow-up; developmental; Development; cost; designing; design; Population; cancer invasiveness; Coupled; innovate; innovative; innovation; prototype; tumor; standard of care; bio-markers; biologic marker; biomarker; Biological Markers; clinical practice; clinical decision-making; screening; Patient risk; diagnostic assay; diagnostic marker; diagnostic biomarker; stratified patient; patient stratification; biomarker discovery; liquid biopsy; Retrospective cohort; Risk stratification; lead optimization; lead candidate

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.

Public Health Relevance Statement:
Project Narrative Early diagnosis is the single most important tool to delay or avoid cancer-related mortality. Many people undergoing diagnostic imaging today are found to have nodules in their thyroid, which in some cases are malignant; however, it is currently very difficult to predict which nodules are true malignant lesions and which will remain benign and asymptomatic. Using our previously validated technology, we have discovered functional biomarkers to differentiate benign from malignant thyroid nodules. In the current proposal we aim to further develop these novel markers into a diagnostic assay, to facilitate early detection of malignancy, as well as to avoid unnecessary and costly surgeries for cases with benign nodules.

Project Terms:
Affect; Antibodies; Autopsy; necropsy; postmortem; Award; Biological Assay; Assay; Bioassay; Biologic Assays; Biological Sciences; Biologic Sciences; Bioscience; Life Sciences; Biopsy; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Malignant neoplasm of thyroid; Malignant Thyroid Gland Neoplasm; Malignant Tumor of the Thyroid; Malignant Tumor of the Thyroid Gland; Thyroid Cancer; Cells; Cell Body; Cytology; Diagnosis; Diagnostic Imaging; Disease; Disorder; Feasibility Studies; Fluorogenic Substrate; Goals; Gold; Indiana; Laboratories; mortality; Persons; Nodule; Nucleotides; Pathology; Patients; Esteroproteases; Peptidases; Protease Gene; Proteases; Proteinases; Proteolytic Enzymes; Peptide Hydrolases; Proteins; Risk; Science; Sensitivity and Specificity; Specificity; Technology; Testing; Thyroid; Thyroid Head and Neck; Thyroid Gland; Thyroid Gland Nodule; Thyroid Nodule; Thyroidectomy; Tissues; Body Tissues; Ultrasonography; Echography; Echotomography; Medical Ultrasound; Ultrasonic Imaging; Ultrasonogram; Ultrasound Diagnosis; Ultrasound Medical Imaging; Ultrasound Test; diagnostic ultrasound; sonogram; sonography; sound measurement; ultrasound imaging; ultrasound scanning; United States; Universities; Work; Measures; Diagnostic tests; Guidelines; base; improved; Benign; Clinical; Malignant - descriptor; Malignant; Phase; Biological; biologic; prognostic; Lesion; enzyme activity; Collaborations; Contracting Opportunities; Contracts; tool; Diagnostic; programs; Unnecessary Surgery; Investigation; Frequencies; Clinic; Source; Best Practice Analysis; Benchmarking; Operative Procedures; Surgical; Surgical Interventions; Surgical Procedure; surgery; Operative Surgical Procedures; early detection; Early Diagnosis; Performance; Protein Cleavage; Proteolysis; Accuracy of Diagnosis; diagnostic accuracy; cohort; Biopsy Sample; Biopsy Specimen; validation studies; Categories; Reporting; Abscission; Extirpation; Removal; Surgical Removal; resection; Excision; Sampling; Proteomics; Genomics; Documentation; Tissue Sample; Preparedness; Readiness; Address; FNA; Fine Needle Aspirate; Fine-Needle Aspiration; Fine needle aspiration biopsy; CHTN; Cooperative Human Tissue Network; DNA Alteration; DNA mutation; Genetic mutation; Sequence Alteration; genomic alteration; DNA Sequence Alteration; Data; Detection; Molecular Analysis; Predictive Value; precancerous; premalignant; Clinical Management; Genetic Risk; Risk Marker; Scientific Advances and Accomplishments; scientific accomplishments; scientific advances; Validation; Pathologic; Monitor; Molecular; Resected; Development; developmental; Image; imaging; cost; Prevalence; cancer invasiveness; prospective; innovation; innovate; innovative; commercialization; standard of care; novel marker; new marker; novel biomarker; Biological Markers; bio-markers; biologic marker; biomarker; clinical practice; biobank; biorepository; clinical decision-making; Institutional Review Boards; IRB; IRBs; precision medicine; precision-based medicine; Patient risk; diagnostic assay; Industry Standard; diagnostic biomarker; diagnostic marker; patient stratification; stratified patient; biomarker discovery; Prospective cohort; Retrospective cohort; risk stratification; stratify risk; clinical center; rapid test; rapid assay; rapid tests; detection limit; ultrasound; diagnostic tool