Supplemental Materials. Supplemental Table 1: Patient mutational status and agreement between qPCR, dPCR and sequencing results. Supplemental Table 2: Analysis of different tissue samples coming from the same patient. Supplemental Figure 1: Fraction of mutated alleles (including multiple subclones) in the patients detected by both conventional and droplet-digital PCR procedures. Supplemental Figure 2: Fraction of mutated DNA observed for the different tested mutations of KRAS and BRAF.

Background: In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence. Method: The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3-4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy. Results: Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71-7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32-7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1-6 months) (adjusted HR = 5, 95% CI 1.9-12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study. Conclusion: This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques. (C) 2021 Elsevier Ltd. All rights reserved.

An azobenzene-core chiral mesogen designed for a photoactive ferroelectric liquid crystal system with switchable polarisation displays a highly unusual phase sequence, with a re-entrant, optically isotropic, fluid phase found below smectic phases in mixtures with high enantiomeric purity. The re-entrant isotropic phase is found on the basis of X-ray scattering and freeze-fracture transmission electron microscopy experiments not to be a cubic or other highly ordered phase but instead a translationally disordered liquid. The material also forms a gel under a wide range of concentrations in 50: 50 ethanol/chloroform solutions. Ultraviolet/visible and infrared spectroscopy and quantum chemistry calculations suggest that the primary unit in the re-entrant isotropic and gel phases is a dimer composed of molecules crossed by about 90 degrees, which hinders the formation of crystal phases and forms tubules of helical aggregates in the gel phase.

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

622 Background: Circulating tumor DNA (ctDNA) is thoroughly investigated as a surrogate biomarker of tumor follow-up, in different cancer types, such as colorectal cancer (CRC). Droplet-based digital PCR (ddPCR) is a highly sensitive and also quantitative method for detection of very low amount of ctDNA. Since many different genes can be mutated within a specific tumor type and also wide mutation spectrum can occur within a specific gene, procedures for ctDNA monitoring can be time consuming and need to be improved for a routinely use. To overcome these drawbacks, we characterized the methylation status of 3 genes frequently hypermethylated in CRC to identify universal markers for tumor follow-up. Methods: The characterization of the methylated status of the WIF, NPY and PENK genes in the tumor DNA was performed in 56 CRC of different stages and 45 corresponding plasma samples using droplet-based dPCR, after DNA bisulfite conversion. A two-panels assay (with albumin as a reference) was developed. Methylation level of these 3 genes in tumor tissues was compared to corresponding normal tissues (n = 22) and plasma samples (MetctDNA). To validate, plasma samples of additional 91 patients were analyzed for the presence of ctDNA both by the characterization of KRAS, BRAF, TP53 and PIK3CA mutations (MutctDNA) and of MetctDNA, at various stages of their follow-up, and 9 of them had MetctDNA assessment during treatment follow-up. Results: All tumor samples were positive for WIF1 and/or NPY markers. Hypermethylation of these two genes was significantly higher in tumor tissue compared to normal, independently of the tumor stage (p < 0.0001). MetctDNA could be detected in 75% of metastatic CRC patients and 24% of localized CRC patients (stage 1 to 3). MetctDNA and MutctDNA fractions were strongly correlated (R2 > 0.9, p < 0.0001). During follow-up, MetctDNA levels changes allowed monitoring of tumor evolution in different stages CRC patients. Conclusions: These results indicate that determination of MetctDNA by droplet-based dPCR can reach same efficiency than MutctDNA for ctDNA assessment, using only 2 markers, and thus could be considered as a universal surrogate marker of tumor follow-up in CRC patients.

Abstract Purpose: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis. Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction. Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors. Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies. Clin Cancer Res; 21(5); 1087–97. ©2014 AACR.

Abstract Circulating tumor DNA has been suggested to be a prognostic factor in different types of tumors. In order to investigate this role, we prospectively enrolled 250 patients with stage II and stage III colorectal cancer (NCT01198743). Plasma samples were collected before surgery (D0) and 5 days later (D5), then every 4 months during the 3 years of follow-up. The end of follow-up will be May 2017. The primary tumors were characterized for KRAS, NRAS, BRAF, PIK3CA, APC and TP53 mutation by sequencing. The circulating DNA was assessed by picoliter digital droplet PCR (dPCR) using the Raindrop instrument (Raindance technologies) with Taqman probes for the 7 most frequent mutations of KRAS and for the BRAF V600E mutation. The survival without recurrence curves were estimated with the Kaplan- Meier method. Differences between groups of patients were analyzed using unstratified log-rank tests. For comparisons of baseline characteristics, categorical outcomes were analyzed with χ2 tests and continuous outcomes were compared with standard parametric or nonparametric tests. Continuous variables are presented as the mean (SD) and median interquartile rang. We present here the results of tumor circulating DNA for the first 56 patients with KRAS or a BRAF mutated tumors. The mean age was 65.3±11 years [37-84], the gender ratio M/F was 0.9. The tumor location was proximal or distal in 55% and 45% respectively. TNM classification showed 49% and 51% of stage II and stage III respectively. The tumors were found mutated for BRAF in 9 cases (16%) and mutated for KRAS in 47 cases (84%). Finally 54% of the patients received an adjuvant chemotherapy. Tumor circulating DNA was found in 12 patients out of the 56 tested patients (23%). A mutation identical to that found in the tumor was identified at D0 or D5 after surgery before any chemotherapy treatment in the plasma of 12 patients (21.4%). For the patients with circulating tumor DNA, the mutated allele frequency in the plasma range from 0.2‰ to 1.4% (median 2.4‰). The detection of circulating tumor DNA was more frequent in patients with recurrence or death from the disease during the follow-up than those without at the time of diagnosis (45.4% versus 15.6%, p = 0.03). Patients without circulating tumor DNA before or 5 days after surgery have a significant better survival without recurrence than those with circulating tumor DNA detectable in univariate analysis (HR: 7.3 CI95% [1.7-31.5] p = 0.006). After adjustment on tumor stage this result remains significant (HR: 6.9 CI95% [1.5-32.9] p = 0.01) During the follow-up DNA was detected in 36.7% of patients who recurred or died from the disease as compared to 2.2% of those who do not (P = 0.004). These preliminary results showed a strong prognostic impact of circulating tumor DNA at the time of surgery and need to be confirmed in the whole series of 250 patients and with the complete follow-up of 5 years. Citation Format: Pierre Laurent-Puig, Olivier Bouché, Ralph Niarra, Pascaline Aucouturier, Leonor Benhaim, Bruno Landi, Anne Berger, Thierry Lecomte, Corinne Normand, Delphine Le Corre, Audrey Didelot, Karine Mallet, Karla Perez Toralla, Thevy Hor, Zakaria El Harrak, Gilles Chatellier, Brian Hutchison, Darren Link, Valerie Taly. Circulating tumor DNA as a prognostic marker in colorectal cancer: Preliminary results of a prospective trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2015-5235

Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.

Abstract Alongside conventional chemotherapies, targeted treatments have significantly improved the survival of patients with metastatic colorectal cancer (mCRC) from 6 months with best supportive care treatment, to 24 months with a combination of conventional and targeted chemotherapies. However, patients with KRAS mutated tumors have little or no benefit from these anti-EGFR antibodies based therapies (Panitumumab, Cetuximab) as single agents or combined with chemotherapy. These findings led to an amendment of the Marketing Authorization of these drugs that are now restricted to patients with KRAS wild-type tumor. However, the response of these patients ranged from 40 to 60%. Recent works have demonstrated that secondary resistance to such therapies was associated with emergence of KRAS mutated subclones in KRAS wild-type patients at the diagnosis time. To understand the importance of KRAS mutant subclones at the diagnosis time and to evaluate the consequence of their presence in terms of management of mCRC patients, a highly sensitive and quantitative procedure is required. Droplet-based digital PCR has recently emerged as a highly sensitive and quantitative approach for rare sequence detection. A retrospective study was set up where tumor samples from 177 mCRC patients were analyzed using a multiplex highly sensitive droplet-based procedures and ultra-deep sequencing using a new droplet-based target enrichment. Results are compared with data obtained by conventional qPCR. Sixty-two patients were classified as responders according to RECIST criteria. All tumors (41) detected as positive using conventional procedures were also positive with our procedure. Among the samples detected as negative with conventional procedures, 23 presented a KRAS or a BRAF mutation only detected by droplet-based procedures and 6 presented additional subclones. For 20 patients, additional biopsy samples were available and presented comparable results. 167 samples were also submitted to deep sequencing and, for alleles detected at fractions superior to 1%, the mutational status of the sample were in agreement for 94% of the samples (157/167) and we observed a correlation with the observed fraction of mutant alleles with the two procedures (R2= 0.7). We observed an inverse correlation between the proportion of mutated DNA and the frequency of anti-EGFR responses (P< 0.0001). The mean percentage of mutated DNA was 0.45% and 12.7% for responders and non-responders respectively. Progression Free Survival of patients was significantly different depending on the percentage of KRAS mutated allele within the tumor. The Progression Free Survival of patients with tumor presenting less than 5% of mutated KRAS was comparable to the one of non-mutated patients. HRs were of 1.07 (CI95% 0.6-2, NS), 2.9 (CI95% 1.7-4.9, P<0.001), 3.6 (CI95% 2.2-5.8, P<0.001) for patients with tumor containing less than 5%, between 5 and 25% or more than 25% of mutated DNA respectively. Citation Format: Valerie Taly, Pierre Laurent-puig, Deniz Pekin, Corinne Normand, Steve K. Kotsopoulos, Philippe Nizard, Jeff Olson, Preethi Srinivasan, Delphine Le Corre, Xinyu Li, Qun Zhong, Darren R. Link, Olivier Bouché, Jean-François Emile, Bruno Landi, Valérie Boige, Brian J. Hutchison. Clinical significance of low frequency KRAS and BRAF subclones for advanced colon cancer management. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2014-2820