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Prospective longitudinal liquid biopsy study

Dynamic circulating tumour cell phenotypes and ctDNA evolution during PARP inhibitor therapy in HGSOC

Functional CTC heterogeneity and longitudinal molecular evolution in HGSOC

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Can liquid biopsy reveal emerging treatment resistance before clinical relapse?

CTCs Circulating DNA
One blood sample · two complementary readouts

PARP inhibitor · schematic

HGSOC is a heterogeneous disease

High-grade serous ovarian cancer (HGSOC) contains diverse tumour subclones. Differences in DNA repair can shape how they respond to treatment.

PARP inhibition applies pressure

PARP inhibition exploits homologous recombination deficiency (HRD), making cells with impaired DNA repair more vulnerable to treatment.

Treatment-sensitive cells respond

As treatment-sensitive cells decline, imaging and CA-125 can reflect the clinical response.

A subpopulation may persist

Some tumour cells may survive treatment. Months can pass before clinical relapse, leaving their survival during therapy poorly characterised.

Objective

To longitudinally characterise functional CTC phenotypes and ctDNA molecular evolution during PARPi maintenance therapy.

Study design

Eleven patients, followed through treatment

A prospective longitudinal cohort of 11 patients with advanced HGSOC receiving PARPi maintenance therapy, with peripheral blood collected at three points.

PARP inhibitor maintenance therapy

Baseline 6 weeks 12 months

Cellular readout

Circulating tumour cells

Isolated, enumerated, and functionally characterised by PARP1 and γH2AX expression.

Molecular readout

Circulating tumour DNA

Tempus xF 105-gene cfDNA next-generation sequencing panel, in patients with sufficient plasma availability.

Sequential analysis

CTC burden, phenotypic profiles, variant allele frequency (VAF) and molecular evolution during treatment.

One blood sample. Two complementary windows into tumour evolution.

Per-patient CTC counts

0 5 10 15 20 25 30 CTC count Baseline 6 weeks 12 months n = 10n = 10n = 8 Patient HSA-01 · Relapse Patient HSA-01 · Baseline: 0 CTCs Patient HSA-01 · 6 weeks: 0 CTCs Patient HSA-01 · 12 months: 16 CTCs Patient HVN-01 · No relapse Patient HVN-01 · Baseline: 7 CTCs Patient HVN-01 · 6 weeks: 1 CTCs Patient HVN-01 · 12 months: 1 CTCs Patient HVN-02 · Relapse Patient HVR-01 · Relapse Patient HVR-01 · Baseline: 1 CTCs Patient HVR-01 · 6 weeks: 0 CTCs Patient HVR-01 · 12 months: 6 CTCs Patient HVR-02 · Relapse Patient HVR-02 · Baseline: 9 CTCs Patient HVR-02 · 6 weeks: 0 CTCs Patient HVR-02 · 12 months: 4 CTCs Patient HT-01 · Relapse Patient HT-01 · Baseline: 0 CTCs Patient HT-01 · 6 weeks: 0 CTCs Patient HT-02 · No relapse Patient HT-02 · Baseline: 8 CTCs Patient HT-02 · 6 weeks: 3 CTCs Patient HT-02 · 12 months: 0 CTCs Patient HT-03 · No relapse Patient HT-03 · Baseline: 0 CTCs Patient HT-03 · 6 weeks: 0 CTCs Patient HT-03 · 12 months: 21 CTCs Patient HT-04 · No relapse Patient HT-04 · Baseline: 4 CTCs Patient HT-04 · 6 weeks: 4 CTCs Patient HT-04 · 12 months: 0 CTCs Patient HT-05 · Relapse Patient HT-05 · Baseline: 26 CTCs Patient HT-05 · 6 weeks: 0 CTCs Patient HJ-01 · Relapse Patient HJ-01 · Baseline: 0 CTCs Patient HJ-01 · 6 weeks: 0 CTCs Patient HJ-01 · 12 months: 0 CTCs
Relapse No relapse
View per-patient counts
Per-patient CTC counts supplied by the study author. — indicates an unavailable measurement; 0 is a measured zero.
Patient Outcome Baseline6 weeks12 months
HSA-01 Relapse 0016
HVN-01 No relapse 711
HVN-02 Relapse ———
HVR-01 Relapse 106
HVR-02 Relapse 904
HT-01 Relapse 00—
HT-02 No relapse 830
HT-03 No relapse 0021
HT-04 No relapse 440
HT-05 Relapse 260—
HJ-01 Relapse 000

Baseline

60.0%

CTCs were detected in 60.0% of evaluable patients, with marked interpatient variability.

6 weeks

An early decrease in CTC counts was observed after 6 weeks of PARPi treatment.

12 months

CTCs persisted or re-emerged at 12 months in a subset of relapsing patients, suggesting dynamic biological adaptation.

PARP1⁺ / γH2AX⁻

PARP1 POSITIVE

The CTCs expressed PARP1 — the enzyme the therapy is designed to inhibit was present in the cells still circulating.

γH2AX NEGATIVE

But γH2AX, the marker of DNA double-strand breaks, was largely absent. γH2AX-positive CTCs were rarely observed during follow-up: these cells were not registering the damage the treatment should be causing.

PARP1⁺ / γH2AX⁻ CTC PHENOTYPE

Patients with this CTC phenotype, by relapse outcome

85.7%

Patients with relapse

25.0%

Patients without relapse

Group denominators are not reported in the abstract.

This phenotype was detected more frequently in patients who developed relapse, potentially compatible with reduced DNA damage response activation.

Preliminary findings from a prospective longitudinal cohort. Associations described here do not establish causality.

TP53 · qualitative findings below. The chart shows BRCA variants only.

0 25 50 75 VAF (%) 50% — germline expectation Baseline 6 weeks 12 months 49.1% 49.0% 50.4% 49.7% 49.9% 47.3%
HT-04 · BRCA1 p.Y655fs HT-05 · BRCA2 p.L2092fs
View VAF values
Reported variant allele frequency (%)
Patient / variantBaseline6 weeks12 months
HT-04
BRCA1 p.Y655fs		 49.1%49.0%50.4%
HT-05
BRCA2 p.L2092fs		 49.7%49.9%47.3%

Two variants, followed across a year

Sequential cfDNA sequencing tracked pathogenic BRCA alterations in patients HT-04 and HT-05 through all three timepoints.

Stable, and close to 50%

Persistent pathogenic BRCA alterations showed stable VAFs close to 50% across sequential samples, supporting their likely germline origin.

Underneath, something moves

In HT-04, low-frequency TP53 variants showed temporal fluctuations or disappearance during follow-up, suggesting somatic clonal dynamics under treatment pressure.

Not shown as a curve: the reported findings for these TP53 variants are qualitative, and no sequential VAFs are available for them.

Two windows onto the same evolving tumour

Function

CTCs

PARP1 / γH2AX

Genome

ctDNA

mutations / VAF

Tumour evolution

PARPi resistance?

The question mark is intentional. These are eleven patients, followed prospectively — enough to describe a pattern, not to establish a mechanism.

  • Longitudinal liquid biopsy enables real-time monitoring of both functional CTC heterogeneity and ctDNA molecular evolution during PARPi treatment.

  • Persistence of PARP1⁺/γH2AX⁻ CTCs may reflect resistant tumour subpopulations associated with relapse.

  • Dynamic changes in low-frequency somatic variants support ongoing clonal evolution during therapy.

Take-home message

Integrating functional CTC phenotyping with sequential cfDNA profiling may provide a minimally invasive strategy for treatment monitoring and identification of resistance mechanisms in HGSOC.