Droplet digital PCR revealed high concordance between primary tumors and lymph node metastases in multiplex screening of KRAS mutations in colorectal cancer

Váňová, Barbora; Kalman, Michal; Jašek, Karin; Kasubova, Ivana; Burjanivová, Tatiana; Farkašová, Anna; Kružliak, Peter; Büsselberg, Dietrich; Lasabová, Zora

doi:10.1007/s10238-019-00545-y

Cited by 20 publications

(8 citation statements)

References 28 publications

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“…Low-sensitivity methods have been demonstrated to detect < 50% of the mutations that can be detected with high-sensitivity methods. 30 In the present study, the median of the mutated allele frequencies found was < 5% for the mutations detected using ultra-deep NGS but not using Sanger sequencing. The heterogeneity of the mutated allele fractions could also explain why 5% of the patients considered to have wild-type KRAS for the primary tumor in a clinical routine setting were found to have KRAS-mutated metastases.…”

Section: Discussioncontrasting

confidence: 55%

High Concordance and Negative Prognostic Impact of RAS/BRAF/PIK3CA Mutations in Multiple Resected Colorectal Liver Metastases

Brunsell

Sveen

Bjørnbeth

et al. 2020

Clinical Colorectal Cancer

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We investigated the intra-patient heterogeneity of driver gene mutations among colorectal liver metastases by sequencing 479 tumor samples from 106 patients. A near-perfect intra-patient concordance was found in the mutation status of the primary tumor and multiple metastatic lesions of KRAS/NRAS/BRAF and PIK3CA when high-sensitivity methods were applied. Mutations in KRAS alone and KRAS/NRAS/BRAF combined had a negative prognostic impact after liver resection. Background: The prevalence and clinical implications of genetic heterogeneity in patients with multiple colorectal liver metastases remain largely unknown. In a prospective series of patients undergoing resection of colorectal liver metastases, the aim was to investigate the inter-metastatic and primary-to-metastatic heterogeneity of mutations in KRAS, NRAS, BRAF, and PIK3CA and their prognostic impact. Patients and Methods: We analyzed the mutation status among 372 liver metastases and 78 primary tumors from 106 patients by methods used in clinical routine testing, by Sanger sequencing, by next-generation sequencing (NGS), and/or by droplet digital polymerase chain reaction. The 3-year cancer-specific survival (CSS) was analyzed using the Kaplan-Meier method. Results: Although Sanger sequencing indicated inter-metastatic mutation heterogeneity in 14 of 97 patients (14%), almost all cases were refuted by high-sensitive NGS. Also, heterogeneity among metastatic deposits was concluded only for PIK3CA in 2 patients. Similarly, primary-to-metastatic heterogeneity was indicated in 8 of 78 patients (10%) using Sanger sequencing but for only 2 patients after NGS, showing the emergence of 1 KRAS and 1 PIK3CA mutation in the metastatic lesions. KRAS mutations were present in 53 of 106 patients (50%) and were associated with poorer 3-year CSS after liver resection (37% vs. 61% for KRAS wild-type; P ¼ .004). Poor prognostic associations were found also for the combination of KRAS/NRAS/BRAF mutations compared with triple wild-type (P ¼ .002). Conclusion: Intrapatient mutation heterogeneity was virtually undetected, both between the primary tumor and the liver metastases and among the metastatic deposits. KRAS mutations separately, and KRAS/NRAS/BRAF mutations combined, were associated with poor patient survival after partial liver resection.

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Section: Discussioncontrasting

confidence: 55%

High Concordance and Negative Prognostic Impact of RAS/BRAF/PIK3CA Mutations in Multiple Resected Colorectal Liver Metastases

Brunsell

Sveen

Bjørnbeth

et al. 2020

Clinical Colorectal Cancer

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“… 28 , 29 By contrast, orthogonal ctDNA detection tests such as ddPCR are designed to detect fewer tumor-specific SNVs at a time, providing less coverage for tumor heterogeneity. 29 , 30 Thus, these differences may explain lower sensitivity achieved with ddPCR. Although these results are encouraging and establish the superior performance of mPCR-NGS–based ctDNA assay over ddPCR and CEA, a validation of this comparison with a larger sample set is warranted.…”

Section: Discussionmentioning

confidence: 99%

Detection of Molecular Residual Disease Using Personalized Circulating Tumor DNA Assay in Patients With Colorectal Cancer Undergoing Resection of Metastases

Loupakis

Sharma

Derouazi

et al. 2021

JCO Precision Oncology

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PURPOSE More than 50% of patients with stage IV colorectal cancer (metastatic colorectal cancer [mCRC]) relapse postresection. The efficacy of postoperative systemic treatment is limited in this setting. Thus, these patients would greatly benefit from the use of a reliable prognostic biomarker, such as circulating tumor DNA (ctDNA) to identify minimal or molecular residual disease (MRD). PATIENTS AND METHODS We analyzed a cohort of 112 patients with mCRC who had undergone metastatic resection with curative intent as part of the PREDATOR clinical trial. The study evaluated the prognostic value of ctDNA, correlating MRD status postsurgery with clinical outcomes by using a personalized and tumor-informed ctDNA assay (bespoke multiple PCR, next-generation sequencing assay). Postresection, systemic therapy was given to 39.2% of the patients at the discretion of the treating physician. RESULTS Postsurgical, MRD positivity was observed in 54.4% (61 of 112) of patients, of which 96.7% (59 of 61) progressed at the time of data cutoff (hazard ratio [HR]: 5.8; 95% CI, 3.5 to 9.7; P < .001). MRD-positive status was also associated with an inferior overall survival: HR: 16.0; 95% CI, 3.9 to 68.0; P < .001. At the time of analyses, 96% (49 of 51) of patients were alive in the MRD-negative arm compared with 52.4% (32 of 61) in the MRD-positive arm. Patients who did not receive systemic therapy and were MRD-negative in the combined ctDNA analysis at two time points had an overall survival of 100%. In the multivariate analysis, ctDNA-based MRD status was the most significant prognostic factor associated with disease-free survival (HR: 5.78; 95% CI, 3.34 to 10.0; P < .001). CONCLUSION This study confirms that in mCRC undergoing resection of metastases, postoperative MRD analysis is a strong prognostic biomarker. It holds promises for being implemented in clinical decision making, informing clinical trial design, and further translational research.

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“…As a matter of fact, the major technical challenges in testing ctDNA are the low fraction of the mutated allele, which is highly variable in different patients, and the broad dynamic range. Consequently, the most recent efforts are focusing on developing highly sensitive methodologies such as ddPCR and Idylla TM testing [23,26,61,62] In our opinion, ddPCR is the most useful procedure in a daily clinical routine compared to Next Generation Sequencing and Idylla TM , due to low costs and time-saving methodologies. Additionally, ddPCR allows a quantitative assessment of the fractional abundance of the mutation.…”

Section: Discussionmentioning

confidence: 99%

A Real-World Application of Liquid Biopsy in Metastatic Colorectal Cancer: The Poseidon Study

Procaccio

Bergamo

Daniel

et al. 2021

Cancers

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Background: First-line decision making is the key to the successful care of mCRC patients and RAS/BRAF status is crucial to select the best targeted agent. In hub centers, a relevant proportion of patients referred from small volume centers may not have standard tissue-based (STB) molecular results available at the time of the first visit (T0). Liquid biopsy (LB) may help circumvent these hurdles. Methods: A monoinstitutional prospective head-to-head comparison of LB versus (vs.) STB testing was performed in a real-world setting. Selection criteria included: mCRC diagnosis with unknown RAS/BRAF status at T0, tumoral tissue archived in external centers, no previous treatment with anti-EGFR. At T0, patients underwent plasma sampling for LB testing and procedure for tissue recovery. RAS/BRAF genotyping was carried out by droplet digital PCR on circulating-tumoral (ct) DNA. The primary endpoint was the comparison of time to LB (T1) vs. STB (T2) results using the Mann–Whitney U test. Secondary endpoints were the concordance between LB and STB defined as overall percent agreement and the accuracy of LB in terms of specificity, sensitivity, positive and negative predictive value. We also performed an exploratory analysis on urinary (u) ctDNA. Results: A total of 33 mCRC patients were included. Mean T1 and T2 was 7 and 22 days (d), respectively (p < 0.00001). T2 included a mean time for archival tissue recovery of 17 d. The overall percent agreement between LB and STB analysis was 83%. Compared to STB testing, LB specificity and sensitivity were 90% and 80%, respectively, with a positive predictive value of 94% and negative one of 69%. In detail, at STB and LB testing, RAS mutation was found in 45% and 42% of patients, respectively; BRAF mutation in 15%. LB results included one false positive and four false negative. False negative cases showed a significantly lower tumor burden at basal CT scan. Concordance between STB and uctDNA testing was 89%. Conclusions: Faster turnaround time, high concordance and accuracy are three key points supporting the adoption of LB in routinary mCRC care, in particular when decision on first-line therapy is urgent and tissue recovery from external centers may require a long time. Results should be interpreted with caution in LB wild-type cases with low tumor burden.

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Droplet digital PCR revealed high concordance between primary tumors and lymph node metastases in multiplex screening of KRAS mutations in colorectal cancer

Cited by 20 publications

References 28 publications

High Concordance and Negative Prognostic Impact of RAS/BRAF/PIK3CA Mutations in Multiple Resected Colorectal Liver Metastases

High Concordance and Negative Prognostic Impact of RAS/BRAF/PIK3CA Mutations in Multiple Resected Colorectal Liver Metastases

Detection of Molecular Residual Disease Using Personalized Circulating Tumor DNA Assay in Patients With Colorectal Cancer Undergoing Resection of Metastases

A Real-World Application of Liquid Biopsy in Metastatic Colorectal Cancer: The Poseidon Study

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