Liquid Biopsy in Head and Neck Cancer
Liquid Biopsy in Head and Neck Cancer
Overall survival of patients with head and neck squamous cell carcinoma (HNSCC) remains
 unsatisfactory due to often advanced clinical stage at diagnosis and high rate of recurrence
 and second primaries. About 75 % of patients with localized HNSCC are expected to show
 circulating tumor DNA (ctDNA) pre-treatment. ctDNA reflects tumor genome and disease burden
 and is termed 'liquid biopsy' (LB) when collected through venous bloodstream. LB has
 potential to assist in early diagnosis of recurrence and progression, and prediction of
 response to targeted therapeutic agents. Increased metabolic activity measured in positron
 emission tomography-computed tomography (PET-CT) is currently the most sensitive technique to
 detect residual cancer or progression of HNSCC after curative treatment. High metabolically
 active tumor volume (MTV) is associated with treatment resistance and shows independent
 prognostic significance. The objective is (i) to investigate whether MTV detected with PET-CT
 correlates to the pattern and amount of genetic alterations in ctDNA of patients with HNSCC
 referred to radio- (chemo)therapy (RT/CRT). Another objective is (ii) to determine
 sensitivity of LB compared to PET/CT in detecting residual tumor 3 months after completion of
 RT/CRT. Third (iii), genetic landscape in LB and fresh tumor samples will be evaluated to
 detect resistance genes and targets for immunotherapy and surveillance post-treatment. This
 prospective study includes 30 patients with stage III/IV HNSCC. Before onset and 3 months
 from RT/CRT, LB is obtained for next-generation DNA sequencing using a commercial platform.
 ctDNA and digital droplet PCR will be quantified and compared to MTV in simultaneously
 acquired PET-CT. The investigators hypothesize that LB could assist or replace PET/CT in
 response monitoring and detection of recurrence after RT/CRT.
Background

 Head and neck squamous cell carcinoma (HNSCC) stands as the sixth most common cancer
 worldwide. The overall 5-year survival is approximately 50 % due to often advanced clinical
 stage at diagnosis, high rate of treatment resistance, and high incidence of second cancers.
 Currently there are no useful biomarkers for surveillance or diagnosis of recurrent HNSCC.

 Liquid biopsy (LB)

 Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA circulating freely in the
 bloodstream. Apoptosis and necrosis due to rapid cell growth and increased cell turnover
 contribute to release of ctDNA in circulation of patients with cancer even without
 disseminated disease. ctDNA reflects tumor genome and may also reflect disease burden. Due to
 these properties and easy accessibility collection of ctDNA through venous blood has been
 termed as 'liquid biopsy'. Approximately 75-90 % of patients with localized or untreated
 HNSCC are expected to show ctDNA pre-treatment.

 Applications of liquid biopsy in HNSCC

 Potential clinical applications of liquid biopsy (LB) are manifold and include diagnosis of
 recurrence or progression, and disease surveillance. While ctDNA contains the same mutations
 present in original tumor, LB can be used to guide targeted therapies if local treatment is
 not feasible. In HNSCC, these features render study of LB attractive since early diagnosis of
 treatment failure increases possibilities for curative approach. ctDNA reflects tumor
 heterogeneity and findings in surveillance samples are more likely to characterize resistant
 subpopulations compared to pre-treatment changes. Recently, immune checkpoint inhibition with
 monoclonal antibodies against programmed cell death (PD-1) have demonstrated activity against
 recurrent or metastatic HNSCC. LB together with evaluation of expression of PD-1 ligand
 (PD-L1) can assess mutational load thus assisting in screening patients who may benefit from
 immunotherapy.

 Metabolic imaging - current standard for detection of recurrent HNSCC

 Increased metabolic activity detected in positron emission tomography-computed tomography
 (PET-CT) is currently the most sensitive technique to detect residual cancer or progression
 of HNSCC after curative treatment. High metabolically active tumor volume (MTV) is associated
 with treatment resistance and shows independent prognostic significance. While PET-CT shows
 excellent sensitivity, it may lead to false positive findings caused by inflammation or other
 conditions having increased cellular metabolic rate. Furthermore, metabolic imaging does not
 elucidate resistance mechanisms nor does it assist in planning of targeted treatments.
 Whether LB could assist or replace PET/CT in response monitoring and detection of recurrence
 remains thus far unknown.

 Study objectives

 i) To investigate whether MTV detected in PET-CT correlates to pattern and amount of genetic
 alterations in ctDNA of patients with HNSCC referred to radiotherapy (RT) or
 chemoradiotherapy (CRT) ii) To determine whether LB is more sensitive than PET/CT in
 detecting residual tumor three months after completion of RT or CRT iii) To evaluate
 exhaustive genetic landscape of patients with locally advanced HNSCC in order to characterize
 resistance or target genes for alternative treatments including (but not limited to)
 immunotherapy, antibodies against epidermal growth factor receptor (EGFR), and hypoxia- and
 virus-activated compounds iv) To validate a novel digital droplet polymerase chain reaction
 (PCR) technique for non-invasive surveillance of patients at high risk for recurrence of
 HNSCC
Inclusion Criteria:

 -  histologically confirmed head and neck squamous cell carcinoma (HNSCC)

 -  WHO performance status 0-2

 -  clinical stage III patients with bulky T3 primary +/- neck metastasis

 -  all stage IV patients

 -  referral to definitive radiotherapy or chemoradiotherapy or multimodality treatment

 Exclusion Criteria:

 -  patients who are not able to sign written informed consent