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Background: The incidence of glioblastoma in the elderly population is increasing as the worldwide population ages. The differential and poorer survival in the elderly population compared to younger patients is partially explained. The present study aimed to investigate the clinical impact of epidermal growth factor receptor EGFR-altered glioblastoma in a real-life elderly glioblastoma population., Patients and Methods: A bicentric and retrospective study was conducted. Patients were 70 years or older and suffering from histomolecularly confirmed glioblastoma. Single nucleotide variants (SNV), amplification, or chromosome 7 polysomy were sought. The primary endpoint was the comparison of overall survival (OS) in patients with or without EGFR alteration. Secondary objectives were to determine other clinical parameters correlated with EGFR alteration status., Results: Seventy-three patients were analyzed: 41.1% had at least one EGFR alteration. The presence of EGFR alteration did not impact overall survival: HR 0.97 [0.6-1.57], p = 0.9; the median overall survival was 6.5 months [5.3-9.3] in the EGFR-altered group versus 7 months [4.5-10] in the EGFR wild-type group, p = 0.75. In multivariate analysis, tumor resection was associated with a significant overall survival improvement: the median OS in the resected group (n = 20) was 11 months [95% CI 7.8-22] versus a median OS of 5.5 months [4.6-7.8] in the unresected group (n = 53), without correlation to EGFR alteration status., Conclusion: In the modern era of molecular characterization and improved treatment modalities, the presence of at least one EGFR alteration did not influence survival outcomes in an elderly population of glioblastoma patients., Competing Interests: Declarations Competing Interests The authors declare no competing interests. Ethical approval All patients provided written informed consent for the use of de-identified tumor material and demographic data for research purposes., (© 2024. The Author(s).)

Objective: To investigate in patients treated for a resectable pancreatic ductal adenocarcinoma [pancreatic adenocarcinoma (PA)], the prognostic value of baseline carbohydrate antigen 19.9 (CA19-9) and circulating tumor DNA (ctDNA) for overall survival (OS), to improve death risk stratification, based on a planned ancillary study from PANACHE01-PRODIGE 48 trial., Background: Biological borderline situation that was first used by the MD Anderson, became a standard practice following the international consensus conference in 2016 to manage PA. Regarding the risk of systemic disease, especially in the setting of "markedly elevated" CA19-9, neoadjuvant therapy is advised to avoid unnecessary surgery, with a risk of early recurrence. To best define biological borderline situations, new biomarkers are needed., Methods: Characteristics at diagnosis and OS were compared between patients with or without ctDNA status available. OS was estimated with the Kaplan-Meier method and compared with a log-rank test. The restricted cubic spline approach was used to identify the optimal threshold for biological parameters for death risk stratification. Univariate and multivariate Cox proportional hazard models were estimated to assess the association of ctDNA status and other parameters with OS., Results: Among the 132 patients from the primary population for analysis in the PANACHE01 -PRODIGE 48 trial, 92(71%) were available for ctDNA status at diagnosis. No selection bias was identified between patients with or without ctDNA status. Fourteen patients (15%) were ctDNA+ and exhibited a higher risk for death [ P = 0.0188; hazard ratio (95% CI): 2.28 (1.12-4.63)]. In the 92 patients with ctDNA status available among the other parameters analyzed, only CA19-9 was statically associated with OS in univariate analysis. Patients with a log of CA19-9 equal or superior to 4.4 that corresponds to a CA19-9 of 80 UI/mL were identified at higher risk for death [ P = 0.0143; hazard ratio (95% CI): 2.2 (1.15-4.19)]. In multivariate analysis, CA19-19 remained independently associated with OS ( P = 0.0323). When combining the 2 biomarkers, the median OS was 19.4 [IC 95%: 3.8-not reached (NR)] months, 30.2 (IC 95%: 17.1-NR) months and NR (IC 95%: 39.3-NR) for "CA19-9 high and ctDNA+ group," "CA19-9 high or ctDNA+ group," and "CA19-9 low and ctDNA- group," respectively (log-rank P = 0.0069)., Conclusions: Progress in the management of potentially operable PA remains limited, relying solely on strategies to optimize the sequence of complete treatment, based on modern multidrug chemotherapy (FOLFIRINOX, GemNabPaclitaxel) and surgical resection. The identification of risk criteria, such as the existence of systemic disease, is an important issue, currently referred to as "biological borderline disease." Few data, particularly from prospective studies, allow us to identify biomarkers other than CA19-9. Combining ctDNA with CA19-9 could be of interest to best define biological borderline situations in PA., Competing Interests: J.P. received 2 research grants for the LIBRARY project: AFRCP grant of €4,000 and a grant from Canceropole Nord Ouest (France) of €20,000. The remaining authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Cervical cancers are mainly caused by an oncogenic HPV. For locally advanced stages, the standard treatment is radio-chemotherapy (RTCT) followed by brachytherapy. Nevertheless, the prognosis remains highly heterogeneous between patients., Objective: We investigated the prognostic value of HPV circulating tumor DNA (ctDNA) in locally advanced cervical cancers alongside that of Squamous Cell Carcinoma Antigen (SCC-A)., Methods: This single-center retrospective study included patients treated in curative intent for an IB3 to IVA squamous cell cervical cancer. Quantification of HPV ctDNA in serum collected at diagnosis was performed using a multiplex digital PCR assay for the simultaneous detection of 8 HPV genotypes., Results: Among the 97 patients included, 76 patients (78.4%) were treated by RTCT, followed by brachytherapy for 57 patients (60%). HPV ctDNA was detected in 59/97 patients at diagnosis (60.8%). This detection was associated with lymph node invasion (p=0.04) but not with tumor stage. A high level of SCC-A at diagnosis was associated with tumor stage (p=0.008) and lymph node invasion (p=0.012). In univariate analysis, better disease-free survival (DFS) was associated with optimal RTCT regimen (p=0.002), exposure to brachytherapy (p=0.0001) and a low SCC-A at diagnosis (continuous analysis, p=0.002). Exploratory analysis revealed that 3/3 patients (100%) whose HPV ctDNA was still detectable at the end of treatment relapsed, while 6/22 patients (27.3%) whose HPV ctDNA was negative at the end of treatment relapsed., Conclusion: HPV ctDNA detection at diagnosis of locally advanced cervical squamous cell carcinomas is frequent and related to node invasion, but not to DFS. The prognostic value of HPV ctDNA detection after treatment warrants specific studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Beaussire-Trouvay, Duhamel, Perdrix, Lévêque, Vion, Rovelet-Lecrux, Sarafan-Vasseur, Di Fiore, Crouzet, Leheurteur and Clatot.)

Background: Liquid biopsy application is still challenging in glioblastoma patients and the usefulness of short-length DNA (slDNA) fragments is not established. The aim was to investigate slDNA concentration as a prognostic marker in unresected glioblastoma patients., Methods: Patients with unresected glioblastoma and treated by radiochemotherapy (RT/TMZ) were included. Plasmas were prospectively collected at three times: before (pre-) RT, after (post-) RT and at the time of progression. Primary objective was to investigate the impact on survival of slDNA concentration [slDNA] variation during RT/TMZ. Secondary objectives were to explore the association between tumor volume, corticosteroid exposition and [slDNA]; and the impact of slDNA detection at pre-RT on survival., Results: Thirty-six patients were analyzed: 11 patients (30.6 %) experienced [slDNA] decrease during RT/TMZ, 22 patients (61.1 %) experienced increase and 3 patients (8.3 %) had stability. Decrease of [slDNA] during RT/TMZ was associated with better outcome compared to increase or stability: median OS, since end of RT, of 13.2 months [11.4 - NA] vs 10.1 months [7.8 - 12.6] and 6.8 months [4.5 - NA], p = 0.015, respectively. slDNA detection at pre-RT time was associated with improved OS: 11.7 months in the slDNA(+) group versus 8.8 months in the slDNA(-) group, p = 0.004. [slDNA] was not associated with corticosteroids exposition or tumor volume. No influence on survival was observed for both whole cfDNA concentration or slDNA peak size., Conclusion: [slDNA] decrease during radiochemotherapy phase is a favorable prognostic marker on OS for unresected glioblastoma patients. Larger and independent cohorts are now required., Trial Registration: ClinicalTrial, NCT02617745. Registered 1 December 2015, https://clinicaltrials.gov/ct2/show/NCT02617745?term=glioplak&draw=2&rank=1., Competing Interests: Declaration of competing interest MF declares income received for research purposes from Servier® company, benefits for interventions from Seagen® and Novocure®, and payment of congress fees from Gilead® and Pfizer®. FC declares benefits for interventions from BMS®, Merck Serono®, MSD®, Gilead®, Astra Zeneca® and Novartis®, and payment of congress fees from Novartis®, Pfizer®, Merck® and Nutricia®. All these conflicts are outside the field of the submitted work. The other authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Circulating tumor DNA (ctDNA) is reported to be promising in localized colorectal cancer (CRC). The present study aimed to retrospectively evaluate the impact of ctDNA in patients with a resected stage II CRC from the PROGIGE 13 trial with available paired tumor and blood samples. A group of recurrent patients were matched one-to-one with nonrecurrent patients according to sex, tumor location, treatment sequence, and blood collection timing. CtDNA was analyzed by digital PCR according to NGS of tumors. Disease-free survival (DFS) and overall survival (OS) were analyzed based on ctDNA, and the risks of recurrence and death were determined. A total of 134 patients were included, with 67 patients in each group. At least one alteration was identified in 115/134 tumors. Postoperative ctDNA was detected in 10/111 (9.0%) informative samples and was detected more frequently in the recurrent group (16.7% versus 1.8%; p = 0.02). The median DFS of ctDNA+ versus ctDNA- patients was 16.8 versus 54 months (p = 0.002), respectively, and the median OS was 51.3 versus 69.5 months (p = 0.03), respectively. CtDNA was associated with recurrence (ORa = 11.13, p = 0.03) and death (HRa = 3.15, p = 0.01). In conclusion, the presence of postoperative ctDNA is associated with both recurrence and survival in stage II CRC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Grancher, Beaussire, Manfredi, Le Malicot, Dutherage, Verdier, Mulot, Bouché, Phelip, Levaché, Deguiral, Coutant, Sefrioui, Emile, Laurent-Puig, Bibeau, Michel, Sarafan-Vasseur, Lepage and Di Fiore.)

Transarterial chemoembolization (TACE) is used to treat patients with unresectable hepatocellular carcinoma (HCC). We evaluated the clinical impact of a-fetoprotein (AFP) and circulating cell-free and tumor DNA (cfDNA and ctDNA) changes around the TACE procedure. Our prospective monocentric study enrolled consecutive patients treated with TACE, with samples collected at baseline (D - 1), Day 2 (D + 2) and 1 month (M + 1) after TACE. cfDNA was quantified by the fluorometric method, and ctDNA was quantified by digital polymerase chain reaction designed for two hotspot TERT mutations. Computerized tomography scans or magnetic resonance imaging were performed at M + 1 every 3 months following TACE and independently reviewed. The objective was to identify thresholds of cfDNA, ctDNA and AFP changes associated with progressive disease (PD) using receiver operating characteristic curves. Thirty-eight patients were included from March 2018 to March 2019. All markers significantly increased from D - 1 to D + 2 (P < .005), and cfDNA and ctDNA significantly decreased from D + 2 to M + 1 (P < .0001). The analysis of changes from D - 1 to M + 1 identified thresholds at +31.4% for cfDNA and 0% for ctDNA that were significantly associated with PD at M + 1 (44.4% [>+31.4%] vs 3.8% [≤+31.4%] and 50.0% [>0%] vs 5.0% [≤0%], respectively). No significant threshold was identified for AFP. Using a score combining cfDNA and ctDNA, the patients were classified into high- or low-risk PD groups at M + 1, with PD rates of 80.0% vs 4.3% (P = .001) and median progression-free survival times of 1.3 vs 10.3 months (P = .002). Our study suggests that cfDNA and ctDNA increases around the TACE procedure and are associated with therapeutic failure., (© 2021 UICC.)

Inflammatory breast cancer (IBC) is an aggressive BC subtype with poor outcomes. A targetable somatic PIK3CA mutation is reported in 30% of IBC, allowing for treatment by PI3Kα-specific inhibitors, such as alpelisib. The aim of this study was to evaluate the detection rate of circulating PIK3CA mutation in locally-advanced IBC (LAIBC) patients harbouring a PIK3CA mutation on initial biopsy. This monocentric retrospective study was based on available stored plasma samples and tumour biopsies at diagnosis from all LAIBC patients treated with neo-adjuvant chemotherapy (NCT) between 2008 and 2018 at the Centre Henri Becquerel. PIK3CA mutations (E542K, E545K, H1047R/L) were assessed by droplet digital PCR (ddPCR) in plasma samples and tumoral tissue at diagnosis. A total of 55 patients were included. Overall, 14/55 patients (25%) had a PIK3CA mutation identified on baseline biopsy (H1047R = 8; H1047L = 3; E545K = 2; E542K = 1). Among them, 11 (79%) patients had enough DNA for circulating DNA analyses, and corresponding circulating PIK3CA mutations were found in 6/11 (55%). Among the 41 patients without PIK3CA mutations on biopsy, 32 (78%) had enough DNA for circulating DNA analysis, and no circulating PIK3CA mutation was identified. Our results revealed no prognostic or predictive value of PIK3CA mutations at the diagnosis of non-metastatic IBC but highlighted the prognostic value of the cfDNA rate at diagnosis. Our study showed that a corresponding circulating PIK3CA mutation was identified in 55% of LAIBC patients with PIK3CA-mutated tumours, while no circulating mutation was found among patients with PI3KCA wild-type tumours., (© 2021. The Author(s).)

Background: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics., Methods: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle., Results: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p < 0.0001). By dichotomising by the median value, cfDNA, ctDNA and CA19-9 were associated with PD, PFS and OS in multivariate analysis. A circulating scoring system (CSS) combining CEA kinetics and baseline CA19-9 and cfDNA values classified patients based on high (n = 58) and low risk (n = 113) of PD and was independently associated with PD (ORa = 4.6, p < 0.0001), PFS (HRa = 2.07, p < 0.0001) and OS (HRa = 2.55, p < 0.0001)., Conclusions: CEA kinetics alone or combined with baseline CA19-9 and cfDNA are clinically relevant for predicting outcomes in mCRC., Trial Registration Number: NCT01212510., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity., Methods: We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection., Results: For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs., Conclusion: Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The clinical implications of plasmatic cell-free and tumor DNA (cfDNA and ctDNA) are challenging in glioblastoma. This prospective study included 52 consecutive newly diagnosed glioblastoma (n = 49) or gliosarcoma (n = 3) patients treated with concomitant temozolomide and radiotherapy (RT-TMZ), followed by a TMZ maintenance phase. Plasma samples were collected at baseline, before RT-TMZ (pre-RT-TMZ) and at the end of adjuvant TMZ, or at the time of progression in cases of progressive disease (PD). The cfDNA concentration was measured with a fluorometric method, and ctDNA was detected using targeted droplet digital PCR. The main objectives were to analyze the associations between cfDNA and ctDNA measurements during the course of treatment with PD and survival. There was a significant decrease in median cfDNA concentration from baseline to pre-RT-TMZ-19.4 versus 9.7 ng/mL (p < 0.0001)-in the entire cohort. In patients with PD, a significant increase in cfDNA concentration from pre-RT-TMZ to time of PD was observed, from 9.7 versus 13.1 ng/mL (p = 0.037), respectively, while no difference was observed for nonprogressive patients. Neither the cfDNA concentration at baseline nor its kinetics correlated with survival. ctDNA was detected in 2 patients (3.8%) and only in gliosarcoma subtypes.Trial registration ClinicalTrial, NCT02617745. Registered 1 December 2015, https://clinicaltrials.gov/ct2/show/NCT02617745?term=glioplak&draw=2&rank=1 .

Background: Endocrine therapy is recommended as a first-line treatment for hormone receptor-positive metastatic breast cancer (HR+MBC) patients. No biomarker has been validated to predict tumor progression in that setting. We aimed to prospectively compare the risk of early progression according to circulating ESR1 mutations, CA-15.3, and circulating cell-free DNA in MBC patients treated with a first-line aromatase inhibitor (AI)., Methods: Patients with MBC treated with a first-line AI were prospectively included. Circulating biomarker assessment was performed every 3 months. The primary objective was to determine the risk of progression or death at the next follow-up visit (after 3 months) in case of circulating ESR1 mutation detection among patients treated with a first-line AI for HR+MBC., Results: Overall, 103 patients were included, and 70 (68%) had progressive disease (PD). Circulating ESR1 mutations were detected in 22/70 patients with PD and in 0/33 patients without progression (p < 0.001). Among the ESR1-mutated patients, 18/22 had a detectable mutation prior to progression, with a median delay of 110 days from first detection to PD. The detection of circulating ESR1 mutations was associated with a 4.9-fold (95% CI 3.0-8.0) increase in the risk of PD at 3 months. Using a threshold value of 25% or 100%, a CA-15.3 increase was also correlated with progression (p < 0.001 and p = 0.003, respectively). In contrast to ESR1, the CA-15.3 increase occurred concomitantly with PD in most cases, in 27/47 (57%) with a 25% threshold and in 21/25 (84%) with a 100% threshold. Using a threshold value of either 25% or 100%, cfDNA increase was not correlated with progression., Conclusion: The emergence of circulating ESR1 mutations is associated with a 4.9-fold increase in the risk of early PD during AI treatment in HR+MBC. Our results also highlighted that tracking circulating ESR1 mutations is more relevant than tracking CA-15.3 or cfDNA increase to predict progression in this setting., Trial Registration: ClinicalTrials.gov, NCT02473120. Registered 16 June 2015-retrospectively registered after one inclusion (first inclusion 1 June 2015).

Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2-7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.

This review is the second part of the workshop on digital PCR (dPCR) proposed by the working group of the French society of clinical biology. The first part of the paper discusses the advantages and limitations of dPCR for the search of different molecular abnormalities such as point mutations, copy number variants, DNA methylation, RNA analysis and a more innovative application, the single-cell dPCR. This synthesis makes it possible to propose a positioning of the dPCR compared to the other available technologies in a medical laboratory. In a second part, the main current applications of the dPCR will be addressed including the oncology of solid tumors and liquid biopsies, oncohematology and the follow-up of hemopathies treatments by hematopoietic stem cell transplantation. We will also detail non-invasive prenatal diagnosis and diagnosis of mosaic genetic disease, using the example of McCune-Albright syndrome. Several French specialists in the field who have implemented these techniques in their laboratory have written these different examples of applications jointly. In summary, this manuscript offers an up-to-date view of the positioning of dPCR in relation to other existing technologies in order to best meet the expectations of precision medicine.

Background: Spinal myxopapillary ependymoma (SP-MPE) is a subgroup of ependymomas in which after initial gross tumor resection, recurrences occur in more than half of the patients. Anaplastic transformation may also occur and contributes to intraneural and extraneural metastatic dissemination. Extraneural metastases from SP-MPE are rare and worsen the prognosis. In this situation, the noninvasive detection of recurrent somatic mutations in the circulating tumor DNA (ctDNA) from plasma is challenging. Telomerase-reverse transcriptase gene promoter (TERTp) mutation has been identified in a subset of ependymomas with aggressive behavior., Case Description: We report on a patient with TERTp mutated SP-MPE presenting with an extraneural anaplastic metastatic dissemination after iterative local recurrences. From the initial SP-MPE to pleural anaplastic lesion, TERTp C228T mutation was present with allele frequency varying from 33% to 39%. Interestingly, TERTp mutation was also detected by droplet digital polymerase chain reaction in the plasma with a frequency of 2.1% at the time of pleural metastases, highlighting that ctDNA is released in plasma of patients suffering from SP-MPE with extraneural metastatic dissemination., Conclusions: Despite the rarity of this evolution, plasmatic liquid biopsy appears to be a useful diagnostic and follow-up tool in a subset of primary brain tumors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes., Methods: We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis., Results: UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods., Conclusions: The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations., (© 2019 American Association for Clinical Chemistry.)

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets., (©2019 American Association for Cancer Research.)

Background: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM)., Methods: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 μM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 μM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment., Results: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours., Conclusion: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

Digital PCR (dPCR) is a 3rd generation technology that complements traditional end-point PCR and real-time PCR. It was developed to overcome certain limitations of conventional amplification techniques, in particular for the detection of small amounts of nucleic acids and/or rare variants. This technology is in a full swing because of its high sensitivity and major applications in various domains such as oncology, transplantation or non-invasive prenatal testing. Consequently, PCRd also has great interest in many areas of medical biology, particularly for clinical applications aiming at detecting and quantifying specific genetic or epigenetic alterations of nucleic acids, even with specimens containing very low concentration of the nucleic acids of interest (e.g. liquid biopsies). However, this technique requires a good training of users and compliance with certain precautions. A lack in such a knowledge can lead to many errors in the conduct of the experiment and the interpretation of the results. In this review, we present the context in which this technology has emerged by describing in particular its principle and the main factors that can influence the quality of the analysis. Then, we propose a number of practical recommendations for the implementation of a test based on dPCR in clinical laboratories with an eye on quality requirements.

Background: Detection of circulating ESR1 mutations is associated with acquired resistance to aromatase inhibitor (AI) in metastatic breast cancer. Until now, the presence of circulating ESR1 mutations at the end of adjuvant treatment by AI in early breast cancer had never been clearly established. In this context, the aim of the present study was to evaluate the circulating ESR1 mutation frequency at the end of adjuvant treatment and after relapse., Methods: This monocentric retrospective study was based on available stored plasmas and included all early breast cancer patients who completed at least 2 years of AI adjuvant treatment and experienced a documented relapse after the end of their treatment. Circulating ESR1 mutations (D538G, Y537S/N/C) were assessed by droplet digital PCR in plasma samples taken at the end of adjuvant treatment, at time of relapse and at time of progression under first line metastatic treatment., Results: A total of 42 patients were included, with a median adjuvant AI exposure of 60 months (range 41-85). No circulating ESR1 mutation was detectable at the end of AI adjuvant therapy. At first relapse, 5.3% of the patients (2/38) had a detectable circulating ESR1 mutation. At time of progression on first-line metastatic treatment, 33% of the patients (7/21) under AI had a detectable circulating ESR1 mutation compared to none of the patients under chemotherapy (0/10). The two patients with a detectable ESR1 mutation at relapse were treated by AI and had an increase of their variant allele fraction at time of progression on first-line metastatic treatment., Conclusions: Circulating ESR1 mutation detection at the end of AI-based adjuvant treatment is not clinically useful. Circulating ESR1 mutation could be assessed as soon as first relapse to guide interventional studies.

If hormone therapy is a key treatment for hormone receptor positive advanced breast cancers, secondary resistance occurs as a rule. Recently, acquired alterations of the ESR1 gene have been identified as a mechanism of resistance on aromatase inhibitor (AI) treatment. The selective pressure by AI exposure during the metastatic setting triggers the emergence of ESR1 activating mutations. In that context, the "liquid biopsy" concept has been used to detect this molecular resistance before progression. Thus, the ESR1 circulating mutation detection will soon be used in daily practice to help monitoring patients on AI treatment and provide an early change for specific therapies that still have to be determined in prospective clinical trials. This review will present the acquired ESR1 mutations, as well as the methods used for their detection in blood and the potential clinical impact of this approach for hormone receptor positive breast cancer management., (Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: In standard pre-analytical conditions, an isolation step is required for circulating tumor DNA (ctDNA) analysis. The need for this step remains unclear with the development of ultrasensitive detection technologies such as digital PCR (dPCR). The aim of our study was to evaluate the ctDNA detection by dPCR platform either directly from plasma (plasma group, PG) or after an isolation step (isolation group, IG)., Methods: We included 17 patients corresponding to a selection of 43 blood samples in metastatic colorectal cancer patients. For each sample, ctDNA was analyzed with or without isolation step (IG and PG, respectively) using KRAS, NRAS and BRAF mutations identified from the tumor tissue. ctDNA detection was performed after a preamplication step using dPCR platform (QuantStudio™ 3D Digital PCR System). ctDNA detection rate and mutant allelic frequencies (MAF) were compared between IG and PG., Results: Our results showed a detection rate at 93% in IG vs. 88% in PG. The concordance rate between the two groups was 91% (39/43) for ctDNA detection with the four discordant cases occurring in patients with low MAF (<0.5%). The mean value of MAF were 16.9±18.9 and 18.5±18.9 for IG and PG, respectively (p=0.24). The correlation coefficient r 2 for MAF was 0.82 between the two methods (p<0.0001)., Conclusion: In conclusion, our results show that direct detection of ctDNA from unpurified plasma is a feasible approach, particularly from sample with high MAF (>0.5%)., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: The direct comparison of CA19.9, circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) using endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has never been performed for the diagnosis of solid pancreatic tumours (SPTs)., Methods: We included 68 patients with a SPT referred for EUS-FNA. CTCs were analysed using size-based platform and ctDNA using digital PCR. The sensitivity, specificity, negative and positive predictive values were evaluated for each marker and their combination., Results: SPTs corresponded to 58 malignant tumours (52 pancreatic adenocarcinoma (PA) and 6 others) and 10 benign lesions. The sensitivity and specificity for PA diagnosis were 73% and 88% for EUS-FNA, 67% and 80% for CTC, 65% and 75% for ctDNA and 79% and 93% for CA19.9, respectively. The positivity of at least 2 markers was associated with a sensitivity and specificity of 78% and 91%, respectively. CtDNA was the only marker associated with overall survival (median 5.2 months for ctDNA+ vs 11.0 months for ctDNA-, P=0.01)., Conclusions: CA19.9 alone and in combination with ctDNA and/or CTC analysis may represent an efficient method for diagnosing PA in patients with SPTs. Further studies including a larger cohort of patients with both malignant and benign lesions will be necessary to confirm these promising results.

Purpose: Markers of chemotherapy efficacy in metastatic colorectal cancer (mCRC) are essential for optimization of treatment strategies. We evaluated the applicability of early changes in circulating tumor DNA (ctDNA) as a marker of therapeutic efficacy. Experimental Design: This prospective study enrolled consecutive patients with mCRC receiving a first- or second-line chemotherapy. CtDNA was assessed in plasma collected before the first (C 0 ), second (C 1 ) and/or third (C 2 ) chemotherapy cycle, using picodroplet-digital PCR assays based either on detection of gene mutation ( KRAS, BRAF, TP53 ) or hypermethylation ( WIF1, NPY ). CT scans were centrally assessed using RECIST v1.1 criteria. Multivariate analyses were adjusted on age, gender, ECOG performance status (PS), metastatic synchronicity, and treatment line. Results: Eighty-two patients with mCRC treated in first- (82.9%) or second- (17.1%) line chemotherapy were included. Patients with a high (>10 ng/mL) versus low (≤0.1 ng/mL) ctDNA concentration at C 0 had a shorter overall survival (OS; 6.8 vs. 33.4 months: adjusted HR, 5.64; 95% CI, 2.5-12.6; P < 0.0001). By analyzing the evolution of the ctDNA concentration between C 0 and C 2 or C 1 (C 2or1 ), we classified the patients in two groups (named "good" or "bad ctDNA responders"). In multivariate analysis, patients belonging to the group called "good ctDNA responder" ( n = 58) versus "bad ctDNA responder" ( n = 15 ) had a better objective response rate ( P < 0.001), and a longer median progression-free survival (8.5 vs. 2.4 months: HR, 0.19; 95% CI, 0.09-0.40; P < 0.0001) and OS (27.1 vs. 11.2 months: HR, 0.25; 95% CI, 0.11-0.57; P < 0.001). Conclusions: This study suggests that early change in ctDNA concentration is a marker of therapeutic efficacy in patients with mCRC. Clin Cancer Res; 23(18); 5416-25. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Heparin is often used as a blood anticoagulant for tumor marker analysis but results in the inhibition of PCR detection of circulating tumor DNA (ctDNA), which has been deemed a potential "liquid biopsy". We aimed to evaluate the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis., Methods: Plasma samples were collected in heparinized (n=194) and EDTA (n=8) tubes from hormone receptor-positive metastatic breast cancer (HR+MBC) (n=144) and pancreatic adenocarcinoma (PA) patients (n=50). Circulating ESR1 and KRAS mutations were detected with or without heparinase by digital PCR in HR+MBC and PA patients, respectively. Patients were classified into 2 subgroups i) inhibition, I+ and ii) no inhibition, I- based on a threshold of 200copies/μL for PCR inhibition by heparin., Results: In the I+ subgroup (91/144 HR+MBC and 26/50 PA), heparinase treatment significantly improved PCR efficacy, enabling ctDNA detection in 22/91 and 13/26 patients. Moreover, comparable results for ctDNA detection (4/8) were obtained with heparinized and EDTA PA samples. In the I- subgroup, heparinase addition did not quantitatively and qualitatively alter ctDNA detection., Conclusion: Heparinase addition removes the heparin inhibition and allows accurate ctDNA detection in heparinized samples. These findings could make the samples from heparinized blood suitable for ctDNA analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We conducted a prospective study to assess the prognostic impact of selected copy number variations (CNVs) in Stage II-III microsatellite stable (MSS) colon cancer. A total of 401 patients were included from 01/2004 to 01/2009. The CNVs in 8 selected target genes, DCC/18q, EGFR/7p, TP53/17p, BLK/8p, MYC/8q, APC/5q, ERBB2/17q and STK6/20q, were detected using a quantitative multiplex polymerase chain reaction of short fluorescent fragment (QMPSF) method. The primary end-point was the impact of the CNVs on the 4-year disease-free survival (DFS). The recurrence rate at 4 years was 20.9%, corresponding to 14% Stage II patients versus 31% Stage III patients (p < 0.0001). The 4-year DFS was significantly decreased in patients with a loss at DCC/18q (p = 0.012) and a gain at ERBB2/17q (p = 0.041). The multivariate analysis demonstrated that Stage III, a loss at DCC/18q and a gain at ERBB2/17q were independent factors associated with DFS. A combination of DCC/18q and ERBB2/17q was also associated with relapse, with the hazard ratio increasing from 1 to 2.4 (95% confidence interval (CI), 1.5-4.1) and 3.1 (95% CI, 1.2-8.4) in the presence of 0, 1 or 2 alterations, respectively (p = 0.0013). CNVs in DCC/18q and ERBB2/17q are significantly associated with DFS in Stage II-III MSS colon cancer., (© 2016 UICC.)

Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To assess the prognostic and predictive value of circulating ESR1 mutation and its kinetics before and after progression on aromatase inhibitor (AI) treatment., Patients and Methods: ESR1 circulating D538G and Y537S/N/C mutations were retrospectively analyzed by digital droplet PCR after first-line AI failure in patients treated consecutively from 2010 to 2012 for hormone receptor-positive metastatic breast cancer. Progression-free survival (PFS) and overall survival (OS) were analyzed according to circulating mutational status and subsequent lines of treatment. The kinetics of ESR1 mutation before (3 and 6 months) and after (3 months) AI progression were determined in the available archive plasmas., Results: Circulating ESR1 mutations were found at AI progression in 44/144 patients included (30.6%). Median follow-up from AI initiation was 40 months (range 4-94). The median OS was decreased in patients with circulating ESR1 mutation than in patients without mutation (15.5 versus 23.8 months, P=0.0006). The median PFS was also significantly decreased in patients with ESR1 mutation than in patients without mutation (5.9 vs 7 months, P=0.002). After AI failure, there was no difference in outcome for patients receiving chemotherapy (n = 58) versus non-AI endocrine therapy (n=51) in patients with and without ESR1 mutation. ESR1 circulating mutations were detectable in 75% of all cases before AI progression, whereas the kinetics 3 months after progression did not correlate with outcome., Conclusion: ESR1 circulating mutations are independent risk factors for poor outcome after AI failure, and are frequently detectable before clinical progression. Interventional studies based on ESR1 circulating status are warranted.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments.

Acquired estrogen receptor gene (ESR1) mutations have been recently reported as a marker of resistance to aromatase inhibitors in hormone receptor positive metastatic breast cancer. We retrospectively considered seven patients treated for metastatic breast cancer with available samples from the primary tumor before any treatment, cryopreserved metastasis removed during progression and concomitant plasmas. All these seven patients were in disease progression after previous exposure to aromatase inhibitors for at least 6 months, and were assessed for ESR1 mutations detection in tumor and circulating DNA. For these patients, Sanger sequencing identified four metastases with clear ESR1 mutation and one possible, whereas digital PCR identified six mutated metastases. Then, under blind conditions and using digital PCR, corresponding circulating ESR1 mutations were successfully detected in four of these six metastatic breast cancer patients. Moreover, in two patients with serial blood samples following treatments exposure, the monitoring of circulating ESR1 mutations clearly predicted disease evolution. In the context of high interest for ESR1 mutations, our results highlight that these acquired recurrent mutations may be tracked in circulating tumor DNA and may be of clinical relevance for metastatic breast cancer patient monitoring., (© 2015 UICC.)

Background: The detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA., Aim: The aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA., Methods: Digital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements., Results: Digital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p<0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (>75% quartile) versus not reached in patients with a low level (<25% quartile) (p=0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p=0.04)., Conclusions: Chip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: The EGFR 3' untranslated region (UTR) harbors a polyadenine repeat which is polymorphic (A13/A14) and undergoes somatic deletions in microsatellite instability (MSI) colorectal cancer (CRC). These mutations could be oncogenic in colorectal tissue since they were shown to result into increased EGFR mRNA stability in CRC cell lines., Methods: First, we determined in a case control study including 429 CRC patients corresponding to different groups selected or not on age of tumor onset and/or familial history and/or MSI, whether or not, the germline EGFR A13/A14 polymorphism constitutes a genetic risk factor for CRC; second, we investigated the frequency of somatic mutations of this repeat in 179 CRC and their impact on EGFR expression., Results: No statistically significant difference in allelic frequencies of the EGFR polyA repeat polymorphism was observed between CRC patients and controls. Somatic mutations affecting the EGFR 3'UTR polyA tract were detected in 47/80 (58.8%) MSI CRC versus 0/99 microsatellite stable (MSS) tumors. Comparative analysis in 21 CRC samples of EGFR expression, between tumor and non malignant tissues, using two independent methods showed that somatic mutations of the EGFR polyA repeat did not result into an EGFR mRNA increase., Conclusion: Germline and somatic genetic variations occurring within the EGFR 3' UTR polyA tract have no impact on CRC genetic risk and EGFR expression, respectively. Genotyping of the EGFR polyA tract has no clinical utility to identify patients with a high risk for CRC or patients who could benefit from anti-EGFR antibodies.

Biallelic and heterozygous mutations of the BUB1B gene have been reported in mosaic variegated aneuploidy (MVA), a rare disorder characterized by constitutional mosaic aneuploidies associated to severe intrauterine growth retardation, microcephaly and, in most cases, to premature chromatid separation (PCS), highlighting the key role of human BUBR1 in chromosome segregation. To study the consequences of gradual reduction of the BUBR1 protein levels, inhibition of BUB1B expression in model cells was induced using short hairpin RNAs (shRNAs). We obtained stable shRNA-transduced HeLa cells displaying a gradient of residual BUBR1 protein (8.5, 10, 14, 58, and 77%), mimicking the situation of patients' cells harboring one or two BUB1B mutations. Induction of PCS was detected in all transduced cells and its level was correlated to the decrease of BUBR1. Aneuploidy was clearly detected in cells with residual BUBR1 below 50%. Our data demonstrate that the function of the human BUBR1 protein in the spindle checkpoint is remarkably dosage-dependent and that the biological consequences of BUB1B expression reduction on premature chromatid separation and aneuploidy depend on the residual amount of BUBR1. This provides a biological explanation for the mode of inheritance of PCS, which is dominant, and of MVA, which can be recessive in some families and result from the combination of a null allele associated to a common hypomorphic allele in others.

The most common form of genomic instability observed in colorectal cancer is chromosomal instability (CIN), whose molecular bases remain to be determined. We have previously demonstrated that inactivation in human cells of several components of the Pes1-Bop1 complex (BOP1, GRWD1, PES1, ORC6L, and RPL3), involved in ribosome biogenesis, altered chromosome segregation. To determine the contribution to colorectal tumorigenesis of somatic alterations of genes involved in ribosome biogenesis, we screened 56 primary colorectal cancers, using quantitative multiplex PCR of short fluorescent fragments, a sensitive method for the detection of gene dosage alterations. We found that dosage increase of the BOP1 gene was a frequent event, being detected in 39% of the tumors, and we show that it is associated with an increase of BOP1 mRNA. Scanning of 8q24, on which BOP1 is located, revealed that in colorectal cancers, gene dosage increase of BOP1 can be independent from that of MYC and was more frequent than that affecting MYC. Finally, transient overexpression of BOP1 in human cells increased the percentage of multipolar spindles. Together with our previous results, the present study strongly suggests that deregulation of the BOP1 pathway contributes to colorectal tumorigenesis., ((c) 2006 Wiley-Liss, Inc.)

The Rapp-Hodgkin syndrome (RHS, MIM 129400) corresponds to a rare form of anhydrotic ectodermal dysplasia, which shares some features with the ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome (EEC, MIM 604292) resulting from TP63 mutations. We report here, in two unrelated patients with RHS, the identification of two distinct TP63 mutations, corresponding to a novel frameshift mutation (1709DelA, exon 14) located downstream the sterile alpha motif (SAM) domain and to a missense mutation (R279H, exon 7) within the DNA binding domain. Functional analysis of the R279H mutation, which had previously been reported in several EEC families, shows that this mutation disrupted the dominant negative activity of the DeltaNp63alpha and gamma isoforms on the transcriptional activity of TP53. This report shows, on a molecular basis, that RHS is also an EEC-like syndrome resulting from mutations of the TP63 gene, and highlights the wide phenotypic spectrum associated to TP63 mutations.

To identify genes which overexpression results into chromosomal instability (CIN), we developed a biological approach based on a yeast indicator strain in which CIN can be detected by a sectoring phenotype. Screening in this strain of a yeast genomic library cloned into a high copy vector led us to identify, among the clones generating 100% of sectoring colonies, Clb5, one of the six B-type cyclins present in yeast. Overexpression of cyclin B2 and cyclin B1, the two human homologs of Clb5, in the CIN indicator strain resulted also into a sectoring phenotype and induced, like overexpression of Clb5, an abnormal sensitivity to benomyl, indicating that overexpression of B-type cyclins alters the spindle checkpoint. In a series of 38 primary colorectal cancers, we detected in five tumors (13%) an accumulation of cyclin B1, which was neither related to mRNA overexpression nor to mutation within the coding region, and in five other tumors (13%) a 2-10-fold increase of cyclin B2 mRNA which was not related to gene amplification. These results suggest that overexpression of cyclins B, resulting from different mechanisms, could contribute, through an alteration of the spindle checkpoint, to the chromosomal instability observed in cancer.