# Custom assays for personalized, longitudinal monitoring of circulating tumor DNA using PIPSenSeq

> **NIH NIH R44** · FLUENT BIOSCIENCES INC. · 2021 · $825,632

## Abstract

PROJECT SUMMARY / ABSTRACT
Targeted DNA sequencing of cell-free tumor DNA (circulating tumor DNA, ctDNA) shed into a patient’s blood
holds great promise for detection, diagnosis, and monitoring of cancer. Given that all tumor cells have the
potential to shed ctDNA, targeted assays using modern next-generation sequencing methods and digital PCR
can provide insight into the entirety of a patient’s tumor burden. ctDNA is therefore an attractive biomarker for
diagnosis and monitoring of cancer patients via non-invasive peripheral blood draw. Such assays, however,
require ultra-sensitive sequencing fidelity, flexible target multiplexing, and uniform target amplification for
efficient, quantitative, and cost-effective testing. In addition, several challenges arise in the implementation of
assays to identify and monitor tumor-specific mutations extracted from blood. First, the mutant DNA from
cancer cells is rare compared to the background of normal DNA, requiring detection sensitivities below 0.1%
for some clinical applications. Second, cell-free DNA extracted from plasma is limited in mass (<100 ng / 5 ml
sample), and typically degraded to very short fragment lengths, requiring efficient capture and amplification of
the targets of interest. Third, cancer diagnosis and monitoring may require surveillance of a multitude of tumor
or patient-specific mutations, requiring multiplex amplification for efficient sample utilization.
In collaboration with our academic partners at Boston University and the University of California San Francisco,
we have developed a novel next-generation sequencing sample preparation platform that addresses these
critical challenges - PIPSenSeq (Pre-templated Instant Partitions for Sensitive Sequencing). This approach
takes advantage of molecular indexing for consensus-read sequencing in combination with single-molecule
amplification in Poisson-distributed nanoscale partitions. Furthermore, PIPSenSeq provides a simple, rapid
library preparation that does not require complex, expensive instrumentation or microfluidic consumables. In
this proposal we will develop PIPSenSeq as a commercial-ready platform for cancer monitoring, and
demonstrate clinical utility in a study of 60 - 70 head and neck squamous cell carcinoma patients. These
patients will be monitored post-operatively for tumor recurrence using personalized PIPSenSeq panels on
longitudinally collected ctDNA samples.

## Key facts

- **NIH application ID:** 10254378
- **Project number:** 5R44CA244049-03
- **Recipient organization:** FLUENT BIOSCIENCES INC.
- **Principal Investigator:** Kristina Fontanez
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $825,632
- **Award type:** 5
- **Project period:** 2020-09-04 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10254378

## Citation

> US National Institutes of Health, RePORTER application 10254378, Custom assays for personalized, longitudinal monitoring of circulating tumor DNA using PIPSenSeq (5R44CA244049-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10254378. Licensed CC0.

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