Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.
ABSTRACT: During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR).TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%.Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.
Project description:The use of non-invasive biomarkers such as circulating tumor DNA (ctDNA) in head and neck tumors may be of relevance in early diagnosis and eventually improved outcome. We evaluated two different approaches from two case series in Europe and South America including (i) targeted screening of ctDNA mutations, and (ii) detection of TP53 mutations in plasma and oral rinses without previous knowledge of mutational status in tumor samples. Targeted sequencing in 5 genes identified ctDNA mutations in plasma among 42% of HNSCC cases, 67% of who were early stage cases. No association was found between ctDNA mutation detection and overall survival. Sequencing of the entire coding region of the TP53 gene resulted in identification of TP53 mutations in 76% of tumor cases. However, concordance of mutation detection was low between tumor, oral rinses (11%) and plasma (2,7%) samples. Identification of 5 pathogenic TP53 mutations in oral rinses from 3 non-cancer controls gives additional evidence of mutation occurrence in individuals without a diagnosed cancer and presents an additional challenge for the development of ctDNA diagnostic assays.
Project description:<h4>Background</h4>Currently, there is no reliable blood-based marker to track tumor recurrence in endometrial cancer (EC) patients. Liquid biopsies, specifically, circulating tumor DNA (ctDNA) analysis emerged as a way to monitor tumor metastasis. The objective of this study was to examine the feasibility of ctDNA in recurrence surveillance and prognostic evaluation of high-risk EC.<h4>Methods</h4>Tumor tissues from nine high-risk EC patients were collected during primary surgery and tumor DNA was subjected to next generation sequencing to obtain the initial mutation spectrum using a 78 cancer-associated gene panel. Baseline and serial post-operative plasma samples were collected and droplet digital PCR (ddPCR) assays for patient-specific mutations were developed to track the mutations in the ctDNA in serial plasma samples. Log-rank test was used to assess the association between detection of ctDNA before or after surgery and disease-free survival.<h4>Results</h4>Somatic mutations were identified in all of the cases. The most frequent mutated genes were PTEN, FAT4, ARID1A, TP53, ZFHX3, ATM, and FBXW7. For each patient, personalized ddPCR assays were designed for one-to-three high-frequent mutations. DdPCR analysis and tumor panel sequencing had a high level of agreement in the assessment of the mutant allele fractions in baseline tumor tissue DNA. CtDNA was detected in 67% (6 of 9) of baseline plasma samples, which was not predictive of disease-free survival (DFS). CtDNA was detected in serial post-operative plasma samples (ctDNA tracking) of 44% (4 of 9) of the patients, which predicted tumor relapse. The DFS was a median of 9 months (ctDNA detected) versus median DFS undefined (ctDNA not detected), with a hazard ratio of 17.43 (95% CI, 1.616-188.3). The sensitivity of post-operative ctDNA detection in estimating tumor relapse was 100% and specificity was 83.3%, which was superior to CA125 or HE4.<h4>Conclusions</h4>Personalized ctDNA detection was effective and stable for high-risk EC. CtDNA tracking in post-operative plasma is valuable for predicting tumor recurrence.
Project description:Human papilloma virus (HPV)-associated oropharyngeal cancer (OPC) is an independent tumour type with regard to cellular, biological, and clinical features. The use of non-invasive biomarkers such as circulating tumour DNA (ctDNA) may be relevant in early diagnosis and eventually improve the outcomes of patients with head and neck squamous cell carcinoma (HNSCC). Genome-wide discovery using RNA sequencing and reduced representation bisulfite sequencing yielded 21 candidates for methylation-targeted genes. A verification study (252 HNSCC patients) using quantitative methylation-specific PCR (Q-MSP) identified 10 genes (ATP2A1, CALML5, DNAJC5G, GNMT, GPT, LY6D, LYNX1, MAL, MGC16275, and MRGPRF) that showed a significant increase recurrence in methylation groups with OPC. Further study on ctDNA using Q-MSP in HPV-associated OPC showed that three genes (CALML5, DNAJC5G, and LY6D) had a high predictive ability as emerging biomarkers for a validation set, each capable of discriminating between the plasma of the patients from healthy individuals. Among the 42 ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 31 (73.8%), 19 (45.2%), and 19 (45.2%) samples, respectively. Among pre-treatment ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 8/8 (100%), 7/8 (87.5%), and 7/8 (87.5%) samples, respectively. Methylated CALML5, DNAJC5G, and LY6D were found in 2/8 (25.0%), 0/8 (0%), and 1/8 (12.5%) of the final samples in the series, respectively. Here, we present the relationship between the methylation status of three specific genes and cancer recurrence for risk classification of HPV-associated OPC cases. In conclusion, ctDNA analysis has the potential to aid in determining patient prognosis and real-time surveillance for disease recurrences and serves as an alternative method of screening for HPV-associated OPC.
Project description:TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC), with mutations occurring in over two thirds of cases; however, the predictive response of these mutations to cisplatin-based therapy remains elusive. In the current study, we evaluate the ability of the Evolutionary Action score of TP53-coding variants (EAp53) to predict the impact of TP53 mutations on response to chemotherapy. The EAp53 approach clearly identifies a subset of high-risk TP53 mutations associated with decreased sensitivity to cisplatin both in vitro and in vivo in preclinical models of HNSCC. Furthermore, EAp53 can predict response to treatment and, more importantly, a survival benefit for a subset of head and neck cancer patients treated with platinum-based therapy. Prospective evaluation of this novel scoring system should enable more precise treatment selection for patients with HNSCC.
Project description:Head and neck squamous cell carcinomas (HNSCC) form a large heterogeneous group of tumors and have a relatively poor outcome in advanced cases. Revealing the underlying genetic mutations in HNSCC facilitates the development of diagnostic biomarkers, which might lead to improved diagnosis and post treatment surveillance. We retrospectively analyzed mutational hotspots using targeted next-generation sequencing (NGS) of 239 HNSCC tumor samples in order to examine the mutational profile of HNSCC. Furthermore, we assessed prevalence, co-occurrence, and synonymy of gene mutations in (matched) tumor samples. TP53 was found mutated the most frequent with mutation rates of up to 83% in all tumors, compared to mutation rates of between 0 and 21% of CDKN2A, PIK3CA, HRAS, CDK4, FBXW7 and RB1. Mutational co-occurrence predominantly existed between TP53 and PIK3CA, TP53 and CDKN2A, and HRAS and PIK3CA. Mutational synonymy between primary tumor and associated metastasis and recurrence was present in respectively 88% and 89%. TP53 mutations were concordantly mutated in 95% of metastases and in 91% of recurrences. This indicates TP53 mutations to be highly prevalent and concordant in primary tumors and associated locoregional metastases and recurrences. In turn, this provides ground for further investigating the use of TP53 mutations as diagnostic biomarkers in HNSCC patients.
Project description:In cancer patients, circulating tumour-derived DNA (ctDNA) levels imperfectly reflect disease burden apparent on medical imaging. Further evaluation of ctDNA levels over time is needed to better understand the correlation with tumour growth and therapeutic response. We describe ctDNA kinetics within an orthotopic, immunocompetent preclinical rabbit model of local-regionally advanced head and neck squamous cell carcinoma (HNSCC). Monitoring primary tumour and metastatic lymph node volume by computed tomography (CT), we observed a correlation between ctDNA levels and tumour burden. We found that ctDNA detection could precede evidence of tumour on CT. Sensitivity and specificity of ctDNA detection in this model was 90.2% (95% C.I.: 76.9-97.3%) and 85.7% (95% C.I.: 67.3-96.0%), respectively. Rapid tumour growth followed by auto-necrosis and tumour volume contraction produced a spike in ctDNA levels, suggesting that viable tumour cells may be required for sustained ctDNA release. Following surgical resection, both ctDNA and total plasma DNA were correlated with recurrent tumour volume. Our results reveal the complex kinetic behaviour of ctDNA and total plasma DNA upon tumour growth or surgery. This pre-clinical model could be useful for future studies focused on elucidating mechanisms of ctDNA release into the circulation from primary and metastatic sites.
Project description:Over 70% of head & neck squamous cell carcinoma (HNSCC) patients carry TP53 oncogenic mutations. Here we studied the role of specific tumor-derived mutant p53 proteins in the aberrant transcription of long non-coding (lnc) MIR205HG gene in head and neck cancer cells. Methods: To understand the role of lncMIR205HG, that we showed to be transcriptionally regulated by mutant p53 in HNSCC, we have employed siRNA and shRNA in CAL27 and FaDu HNSCC cell lines to suppress p53 gene expression in ChIP assays and RT-qPCR. We validated our findings in a cohort of 522 HNSCC patients from The Cancer Genome Atlas Data Portal (TCGA). We further evaluated our results in 63 HNSCC tumor samples collected at our institute, 32 of which were characterized by mutated TP53 (missense mutations) while 31 were characterized by wild-type TP53. Results: Maturation of pre-MIR205HG transcript produces two non-coding RNAs, lncMIR205HG and hsa-miR-205-5p. Down-regulation of lncMIR205HG expression significantly reduced cell proliferation, cell migration and clonogenic activity of head and neck cancer cells. Expression of MIR205HG was significantly increased in HNSCC with mutated TP53 when compared with matched non-tumoral tissues. Furthermore, MIR205HG expression levels were significantly higher in tumoral samples with mutant p53 than in tumoral tissues expressing wild-type p53. Mechanistically, MIR205HG depletes endogenous miR-590-3p leading to increased cyclin B, cdk1, and YAP protein expression. Conclusions: Taken together, these findings identify a transcriptional and post-transcriptional molecular network that includes mutant p53 protein, lncMIR205HG, YAP, and other proliferation-related genes, which are enriched in HNSCC patients with poor prognosis.
Project description:The potentials of circulating tumour DNA (ctDNA) have been studied for non-invasive disease monitoring in patients with targetable mutations. However, the majority of cancer patients harbour no targetable mutations. A workflow including targeted next-generation sequencing (NGS) and droplet digital PCR (ddPCR) could be used for monitoring treatment in these patients. Thus, our aim was to evaluate the workflow for ctDNA monitoring in a cohort of non-small cell lung cancer patients.Forty patients were prospectively included. Plasma samples were collected prior to and during treatment. NGS (Ion AmpliSeq Colon and Lung Cancer panel v2) was performed on ctDNA from pre-treatment samples. The identified mutations were monitored by ddPCR in consecutively collected samples.Mutations were detected in 21 patients. The most commonly mutated genes were TP53 (N=20) and KRAS (N=13). Treatment was discontinued due to non-response in 18 patients. In 16 of these, a simultaneous increase in ctDNA concentration was observed. A twofold ctDNA concentration increase confirmed in a second successive sample predicted non-response on the following imaging in 83% of patients (10/12).ctDNA monitoring can be used for early detection of non-response in patients without targetable mutations, and therefore could supplement imaging data for treatment monitoring in this subset of patients.
Project description:<h4>Background</h4>TP53 mutation is associated with decreased survival rate in head and neck squamous cell carcinoma (HNSCC) patients. We set out to identify microRNAs (miRNAs) whose expression associates with TP53 mutation and survival in HNSCC.<h4>Patients and methods</h4>We analyzed TP53 status by direct sequencing of exons 2 through 11 of a prospective series of 121 HNSCC samples and assessed its association with outcome in 109 followed-up patients. We carried out miRNA expression profiling on 121 HNSCC samples and 66 normal counterparts. miRNA associations with TP53 mutations and outcome were evaluated.<h4>Results</h4>A TP53 mutation was present in 58% of the tumors and TP53 mutations were significantly associated with a shorter recurrence-free survival. This association was stronger in the clinical subgroup of patients subjected to adjuvant therapy after surgery. The expression of 49 miRNAs was significantly associated with TP53 status. Among these 49, we identified a group of 12 miRNAs whose expression correlates with recurrence-free survival and a group of 4 miRNAs that correlates with cancer-specific survival. The two groups share three miRNAs. Importantly, miRNAs that correlate with survival are independent prognostic factors either when considered individually or as signatures.<h4>Conclusions</h4>miRNAs expression associates with TP53 status and with reduced survival after surgical treatment of squamous cell carcinoma of the head and neck.
Project description:Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.