# The impact of oxygen exposure on clinical biomarkers - an underrecognized source of pre-analytic variability

> **NIH NIH U01** · INDIANA UNIVERSITY INDIANAPOLIS · 2024 · $355,980

## Abstract

When preclinical research fails to replicate human biology, scientific progress stalls, clinical trials falter, and
patients continue to suffer. While many factors contribute to these failures, lack of attention to pre-analytic
variability is a seminal issue. We recently reported that even short-term exposure to ambient air is sufficient to
trigger signaling changes in tumor and non-malignant biospecimens. Those changes in turn alter their biology
and responsiveness to targeted therapies. Oxygen (O2) tension in various organs ranges from 3-9%, whereas
O2 in ambient air is 21%. Using transgenic models of breast cancer and ascitic fluid from patients with ovarian
cancer, we demonstrated differential expression of various signaling molecules including YAP1, NRF2/KEAP1
and WNT/b-CATENIN when biospecimens are collected and processed under physioxia (3-5% O2) compared to
the same biospecimen collected and processed under ambient air. We also observed that basal and drug-
induced signaling networks that determine cellular response to targeted therapies are impacted by exposure to
ambient air. Key signaling molecules affected include pEGFR(Y1068), pPDGFRb(Y751), pAKT(S473),
pERK(T202/Y204), DNMT3A, TET2, and BRD4. Based on these results, we hypothesize that exposure of
biospecimens to ambient air during collection and processing incorrectly estimates the levels of many signaling
molecules that are used as biomarkers to define tumor characteristics and to determine the potential benefit of
targeted therapies. The effects of ambient air exposure during collection and processing extends to
biospecimens from normal tissues, impacting characterization of the cell-of-origin of their associated cancers.
Experiments designed in three aims will test these hypotheses. In the first aim, we will collect breast tumor
biopsies, malignant ascites, and pleural effusions under 1, 3 and 5% O2 reflecting variable O2 tension in tumors,
then perform comparative analysis with and without subsequent exposure to ambient air for signaling molecules
listed above. Since our preliminary studies have shown the effects of ambient air exposure on the levels of
DNMT3A and TET2, two major determinants of DNA methylation, aim 2 will investigate the effects of ambient
air exposure on the DNA methylome and transcriptome. In the third aim, using the unique institutional resource
of Komen Tissue Bank, the world’s only repository of normal breast tissue donated by healthy women to support
research, we will characterize the effects of ambient air exposure on the levels of biomarkers from Aim 1 and
transcriptome. Successful completion of these studies will change how samples are collected and processed,
ensuring that O2 exposure mimics the O2 concentration of the organ to limit the introduction of ambient air O2-
induced changes. In addition, this work will force the development of new collection devices that limit the
exposure of biospecimens to ambient air. With respect to this specific FOA, ...

## Key facts

- **NIH application ID:** 10803677
- **Project number:** 1U01CA278865-01A1
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** Kathy D. Miller
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $355,980
- **Award type:** 1
- **Project period:** 2024-05-15 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10803677

## Citation

> US National Institutes of Health, RePORTER application 10803677, The impact of oxygen exposure on clinical biomarkers - an underrecognized source of pre-analytic variability (1U01CA278865-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10803677. Licensed CC0.

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