Isocitrate dehydrogenase (IDH) variants can lead to the development and/or progression of various solid tumors and hematological malignancies. IDH testing can guide diagnosis, prognosis, and therapeutic choice and typically relies on NGS, IHC, or PCR-based assays. Here, we evaluated the analytical performance of the Idylla IDH1-2 mutation assay for IDH variant detection using 70 fixed samples from patients with solid tumors and 36 DNA extracts from patients with acute myeloid leukemias previously characterized by NGS +/- IHC. Idylla IDH1-2 mutation assay gave 98.1% of valid results with an overall agreement, sensitivity, and specificity of 97.1%, 96.2%, and 98.1%, respectively, compared to NGS. Using commercial DNA standards, the limit of detection of the assay was 1.6% and 0.5% for IDH1 R132H and IDH2 R172K variants, respectively. Based on these data, the Idylla IDH1-2 mutation assay represents a fast and reliable alternative to detect IDH hotspot variants in solid tumors and hematological malignancies using either fixed tissue sections or DNA extracts. Particular attention, however, is needed for the interpretation of cases with cycle of quantification values of the internal controls over 35, for which a variant with low allelic frequency could be missed due to low DNA quantity or quality.

PURPOSE:Modified fluorouracil, leucovorin, irinotecan, and oxaliplatin (mFOLFIRINOX/mFFX) is the standard adjuvant chemotherapy for resected pancreatic ductal adenocarcinoma (PDAC), offering survival benefits over gemcitabine (GEM). However, the contribution of molecular biomarkers to treatment selection remains unclear. Here, we characterize the molecular landscape of tumors from the PRODIGE-24/CCTG PA6 trial and assess the clinical impact of genomic alterations and molecular subtypes. PATIENTS AND METHODS:Tumor DNA sequencing was successfully performed in 317/350 tumors (168 mFFX; 149 GEM), complemented by transcriptomic subtyping using the PurIST classifier. Mutational status of four key PDAC driver genes and 24 homologous recombination repair (HRR)-associated genes was analyzed, alongside single-base substitution (SBS) mutational signatures. Primary and secondary end points were disease-free survival (DFS) and cancer-specific survival (CSS), respectively. RESULTS:In the mFFX group, the PurIST subtype was prognostic, with classical tumors showing superior DFS compared with basal-like tumors (stratified hazard ratio [sHR], 0.48 [95% CI, 0.31 to 0.77]). Among KRAS-mutated patients, mFFX significantly improved DFS compared with GEM (sHR, 0.60 [95% CI, 0.45 to 0.79]; P < .001), while no benefit was observed in KRAS wild-type tumors (interaction test, Pint. = 0.010). HRR and BRCA status were not predictive (Pint. = .568 and Pint. = .785, respectively). The benefit of mFFX was consistent across SBS-positive and SBS-negative subgroups. CONCLUSION:Overall, these results do not support a change in current adjuvant treatment strategies. mFFX remains the standard adjuvant regimen in PDAC, and the observed lack of benefit in KRAS wild-type tumors should be considered hypothesis-generating and warrants further investigation.

Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious BRCA1/2 mutation, these mutations are not the sole cause of HRD. Promoter methylation of BRCA1 and RAD51C contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. BRCA1 and RAD51C promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious BRCA1/2 mutation, and 2 (2.6%) a RAD51C mutation. Methylation analysis revealed BRCA1 promoter methylation in 32 patients (41.6%) and RAD51C promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond BRCA1/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.

The use of poly(ADP-ribose) polymerase inhibitors (PARPi) revolutionized the treatment of BRCA-mutated cancers. Identifying patients exhibiting homologous recombination deficiency (HRD) has been proved useful to predict PARPi efficacy. However, obtaining HRD status remains an arduous task due to its evolution over the time. This causes HRD status to become obsolete when obtained from genomic scars, rendering PARPi ineffective for these patients. Only two HRD tests are currently FDA-approved, both based on genomic scars detection and BRCA mutations testing. Nevertheless, new technologies for obtaining an increasingly reliable HRD status continue to evolve. Application of these tests in clinical practice is an additional challenge due to the need for lower costs and shorter time to results delay. In this review, we describe the currently available methods for HRD testing, including the methodologies and corresponding tests for assessing HRD status, and discuss the clinical routine application of these tests and their technical validation.

Homologous recombination deficiency (HRD) affects 50% of ovarian cancers (OC) and influences PARP inhibitors efficacy. While one-third of HRD tumours harbour a deleterious BRCA1/2 mutation, these mutations are not the sole cause of HRD. Promoter methylation of BRCA1 and RAD51C contributes to 19% and 2% of cases, respectively. A cohort of 224 ovarian cancer (OC) patients tested for HRD validated our droplet digital PCR (ddPCR) technique. DNA was extracted from FFPE tissue, and the Genomic Instability Index (GII) was assessed using the Sophia DDM HRD Solution (Sophia Genetics). BRCA1 and RAD51C promoter methylation was analysed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination (HR) status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harbouring a deleterious BRCA1/2 mutation, and 2 (2.6%) a RAD51C mutation. Methylation analysis revealed BRCA1 promoter methylation in 32 patients (41.6%) and RAD51C promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). Our study confirms HRD extends beyond BRCA1/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for PARP inhibitor therapy.

Radiation therapy (RT) is a mainstay of treatment for a myriad of cancers; however, it remains a controversial option in the management of patients with pancreatic cancer. The prognosis for this disease remains one of the poorest, despite recent advances in chemotherapy, which still has limited efficacy and suffers from multiple forms of resistance. The application of RT in patients with pancreatic cancer remains largely institution and provider dependent. Despite advances in RT where higher doses can be delivered while sparing adjacent normal organs, local control remains a problem, highlighting the necessity for improvements in the current RT approach. Emerging strategies are currently being developed with the aim of improving the effects of RT using “radiosensitization” mechanisms. The objective of radiosensitization is to either enhance DNA damage induced by RT or to prevent its repair or to impair the RT-resistance-associated components of the tumor. The process of radiosensitization can be achieved through the use of conventional chemotherapy agents or by novel molecules that inhibit DNA damage response effectors, as well as cell cycle checkpoints. Additionally, nanoparticles with a high atomic number have the potential to act as radiosensitizers by enhancing the effects of RT specifically in tumor cells. Another avenue of radiosensitization entails the combination of immunotherapy with immune checkpoint inhibitors to increase the immunomodulatory impact of RT. The radiosensitivity of pancreatic ductal adenocarcinoma may also be enhanced by targeting components of the tumor microenvironment or metabolic characteristics associated with resistance to RT. This review aims to provide a comprehensive overview of cutting-edge radiosensitization strategies, from their initial preclinical studies to their current status in clinical trials.

e16454 Background: Analysis of cell-free DNA (cfDNA) is an alternative approach to conventional tumor markers in monitoring solid tumors. Although promising, data about cfDNA utility are lacking in pancreatic cancer, particularly for the follow-up of patients undergoing surgery. The present study aimed to evaluate the role of cfDNA levels and genomic variants detected by next-generation sequencing (NGS) as predictive biomarkers of best outcomes in patients with resected pancreatic ductal adenocarcinoma (PDAC). Methods: This multicentric retrospective study included 15 patients with resectable PDAC. All patients were previously enrolled in the PANCREAS CGE study (NTC02818907). Blood samples were collected at baseline; post-neoadjuvant treatment or prior surgery; post-surgery; and post-adjuvant chemotherapy. cfDNA from 59 plasma samples was quantified and qualified using fluorescence-based assays and sequenced using the Illumina instrument and a bespoke gene-panel. FFPE samples were collected at surgery and analyzed using a 519-gene panel (Nonacus). The detection and tracking of somatic variants in cfDNA was performed., The relationship of variant allele frequency (VAF) and cfDNA concentration with clinical outcomes were investigated. Results: At baseline, patients with low cfDNA concentrations ( < 0.4 ng/µL) demonstrated a significantly higher overall survival (OS) than those with high cfDNA concentrations (44.5 versus 24.3 months; hazard ratio (HR) = 0.154; 95% confidence interval (CI) = 0.02-0.88; p = 0.036, respectively). Following surgery, the median OS was 23.7 and 45.9 months in patients with high and low cfDNA concentrations ( > 1.05 and < 1.05 ng/µL), respectively (HR = 0.170; 95% CI = 0.03-1.44; p = 0.0140). Patients with cfDNA concentration at least twice as high postoperatively as preoperatively exhibited a lower OS than those with stable or decreased concentration during the perioperative period (27.2 versus 45.9 months; HR = 0.2; 95% CI = 0.04-0.97; p = 0.0281). NGS-based detection of variants in cfDNA revealed that increasing VAFs were associated with poorer outcomes. cfDNA concentrations after adjuvant treatment and corresponding variant burden were predictive of disease-free survival (DFS), with a cut-off of 0.25 ng/µL (HR = 0.136; 95% CI = 0.04-0.48; p = 0.0247). Conclusions: This study demonstrates that baseline level of cfDNA concentration and somatic VAFs detected by NGS are independent prognostic factors in PDAC patients. The combination of cfDNA concentrations, VAF dynamics, and variant detection during the perioperative period provides deeper insights into tumor biology and patient prognosis. Additionally, cfDNA concentrations and variant burden following adjuvant treatment are effective in predicting recurrence. These results highlight the application of long-term cfDNA and NGS-based monitoring in clinical practice for PDAC patients as readily available, minimally invasive, and cost-effective biomarkers.

Purpose: Diagnosis and treatment decisions of hormonal breast cancers (BC) are now guided by genomic mutations determination, combined into mutational signatures, and provide insight into the patients’ genomic landscape. This work aims to compare genomic data and signatures extracted from tissue samples collected in the CICLADES study to existing cohorts. Ultimately, the goal is to prove the accuracy of smaller cohorts and provide new relevant data. Materials and methods: DNA from patients of the CICLADES cohort was extracted, sequenced, and custom filtering was applied to the resulting files. Genomic data was pulled from 6 BC cohorts available on cBioPortal.com. In total, 2303 samples were analyzed. Mutational signatures were extracted and matched to known signatures of the Catalogue of Somatic Mutations in Cancer (COSMIC). Tumor Mutation Burden (TMB) and hypermutation were estimated and compared between samples. Results: PIK3CA and TP53 represented the two genes highly mutated across all cohorts. TMB was similar between the CICLADES and CBSM groups, however the MSKCC population showed a significantly higher TMB than both. Nine signatures were extracted, with recurring Single Base Substitutions (SBS) signatures like SBS1, SBS2 and SBS5. The presence of APOBEC-specific signatures was concordant with cohorts presenting APOBEC enrichment. The mean number of mutations was significantly higher in enriched samples for each analyzed cohort. Conclusion: The use of comprehensive genomic profiling provided accurate evaluation of the TMB and extraction of signatures consistent with published literature. The genomic analysis of the tissue samples of the CICLADES cohort brings new and relevant data, comparable to results found in bigger cohorts.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal forms of cancer, with a dismal survival rate. Therapeutic options are restricted in surgery for patients with resectable disease and in chemotherapy for those with unresectable disease. The potential benefits of radiotherapy (RT) or chemoradiotherapy (CRT) have been extensively investigated in both neoadjuvant and adjuvant settings in the management of patients with PDAC. Nevertheless, a substantial number of clinical trials have yielded conflicting findings, thereby rendering the impact of RT on patient survival and margin-negative (R0) resection inconclusive. A comprehensive examination of the historical evolution of RT in PDAC, encompassing the identification of both constraints and opportunities for advancement, is essential to establish RT as a promising therapeutic avenue for patients with PDAC. The aim of this review is to provide a synthesis of past clinical trials and future studies to elucidate the evolving role of RT in the management of PDAC as adjuvant and neoadjuvant CRT across different stages of the disease.

The origin of metastases is a topic that has sparked controversy. Despite recent advancements, metastatic disease continues to pose challenges. The first admitted model of how metastases develop revolves around cells breaking away from the primary tumor, known as circulating tumor cells (CTCs). These cells survive while circulating through the bloodstream and subsequently establish themselves in secondary organs, a process often referred to as the "metastatic cascade". This intricate and dynamic process involves various steps, but all the mechanisms behind metastatic dissemination are not yet comprehensively elucidated. The "seed and soil" theory has shed light on the phenomenon of metastatic organotropism and the existence of pre-metastatic niches. It is now established that these niches can be primed by factors secreted by the primary tumor before the arrival of CTCs. In particular, exosomes have been identified as important contributors to this priming. Another concept then emerged, i.e. the "genometastasis" theory, which challenged all other postulates. It emphasizes the intriguing but promising role of cell-free DNA (cfDNA) in metastasis formation through oncogenic formation of recipient cells. However, it cannot be ruled out that all these theories are intertwined. This review outlines the primary theories regarding the metastases formation that involve CTCs, and depicts cfDNA, a potential second player in the metastasis formation. We discuss the potential interrelationships between CTCs and cfDNA, and propose both in vitro and in vivo experimental strategies to explore all plausible theories.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Therapeutic options for PDAC are primarily restricted to surgery in the early stages of the disease or chemotherapy in advanced disease. Only a subset of patients with germline defects in BRCA1/2 genes can potentially benefit from personalized therapy, with the PARP inhibitor olaparib serving as a maintenance treatment for metastatic disease. Although the role of radiotherapy in PDAC remains controversial, the use of radiosensitizers offers hope for improving cancer management. Previously, we have shown that damage-specific DNA binding protein 2 (DDB2) is a potential prognostic and predictive biomarker for chemotherapy response in PDAC. In this study, we investigated the function of DDB2 in radiotherapy response, with and without radiosensitization by olaparib in PDAC cells. Our findings demonstrated DDB2 resistance to radiation effects, thereby improving cell survival and enhancing the repair of ionizing radiation-induced DNA double-strand breaks. We observed that DDB2 expression enhances the cell cycle arrest in the G2 phase by phosphorylating Chk1 and Chk2 cell cycle checkpoints. Additionally, we identified a novel link between DDB2 and PARP1 in the context of radiotherapy, which enhances the expression and activity of PARP1. Our findings highlight the potential of low-DDB2 expression to potentiate the radiosensitization effect of olaparib in PDAC cells. Collectively, this study provides novel insights into the impacts of DDB2 in the radiotherapy response in PDAC, enabling its employment as a potential biomarker to predict resistance to radiation. Furthermore, DDB2 represents a significant step forward in precision radiotherapy by widening the scope of patients who can be benefiting from olaparib as a radiosensitizer. Hence, this research has the potential to enrich the limited use of radiotherapy in the care of patients with PDAC.

Hormonal BCs are characterized by the expression of hormonal receptors (estrogen or progesterone). They can be treated with endocrine therapy (ET) including anti-aromatase inhibitors (AI) and/or anti-cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. However, such treatments can cause cancer cells to mutate and impact genes such as ESR1, PIK3CA and/or AKT1, which are associated with ET resistance. By establishing the baseline level of these mutations in Formalin-Fixed Paraffin-Embedded (FFPE) DNA from patients with advanced or metastatic BC treated with AI and CDK4/6 inhibitors, the CICLADES-CE study, ancillary to CICLADES trial aims to identify genomic signatures to be monitored during follow-up of clonal evolution. Twenty FFPE samples from female patients diagnosed with advanced BCs and treated with AI were qualified, selected and microdissected. The AllPrep® DNA/RNA FFPE kit (Qiagen) was used to extract DNA. A method of Hybridization Capture-based Target Enrichment with a 516-gene panel was used on the NextSeq 550® (Illumina) for sequencing. Single nucleotide variants, copy number variants and telomere length were detected. Data obtained were then analyzed to identify highly mutated genes, specific mutations in genes of interest and genomic signatures as described by Alexandrov et al, 2013. Among 20 samples, 19 reached the quality criteria to be sequenced. We showed that several genes involved in the PI3-Kinase pathway were mutated across several samples, but no relevant mutations of AKT and ESR1 were found at baseline. Among the samples, 3 genomic signatures were detected, corresponding to validated COSMIC (Catalogue of Somatic Mutations in Cancer) signatures 5, 6 and 30. Those signatures are associated with DNA damage repair and deficiency of the base excision repair system. The signatures found are a mirror of the samples origin and the type of fixation used. With this information we have created a focused gene panel to be used for ctDNA follow-up analysis. It also provides us with a clear baseline mutational landscape for the profiling of the rest of the CICLADES cohort.

The predominant forms of breast cancer (BC) are hormone receptor-positive (HR+) tumors characterized by the expression of estrogen receptors (ERs) and/or progesterone receptors (PRs). Patients with HR+ tumors can benefit from endocrine therapy (ET). Three types of ET are approved for the treatment of HR+ BCs and include selective ER modulators, aromatase inhibitors, and selective ER downregulators. ET is the mainstay of adjuvant treatment in the early setting and the backbone of the first-line treatment in an advanced setting; however, the emergence of acquired resistance can lead to cancer recurrence or progression. The mechanisms of ET resistance are often related to the occurrence of mutations in the ESR1 gene, which encodes the ER-alpha protein. As ESR1 mutations are hardly detectable at diagnosis but are present in 30% to 40% of advanced BC (ABC) after treatment, the timeline of testing is crucial. To manage this resistance, ESR1 testing has recently been recommended; in ER+ HER2− ABC and circulating cell-free DNA, so-called liquid biopsy appears to be the most convenient way to detect the emergence of ESR1 mutations. Technically, several options exist, including Next Generation Sequencing and ultra-sensitive PCR-based techniques. In this context, personalization of ET through the surveillance of ESR1 mutations in the plasma of HR+ BC patients throughout the disease course represents an innovative way to improve the standard of care.

Gene fusions and MET exon skipping drive oncogenesis in 8–9% and 3% of non-small cell lung cancers (NSCLC) respectively. Their detection are essential for the management of patients since they confer sensitivity to specific targeted therapies with significant clinical benefit over conventional chemotherapy. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) account for historical reference techniques however molecular-based technologies (RNA-based sequencing and RT-PCR) are emerging as alternative or complementary methods. Here, we evaluated the analytical performance of the fully-automated RT-PCR Idylla GeneFusion assay compared to reference methods using 35 fixed NSCLC samples. Idylla demonstrated overall agreement, sensitivity and specificity of 100% compared to RNASeq. Interestingly, it succeeded in retrieving 10 out of 11 samples with inconclusive results due to insufficient RNA quality for sequencing. Idylla showed an overall agreement, sensitivity and specificity of 90.32%, 91.67% and 89.47% compared to IHC/FISH respectively. Using commercial standards, the limit of detection of the Idylla system for the most frequent fusions and exon skipping ranges between 5 and 10 ng RNA input. These results support that the Idylla assay is a reliable and rapid option for the detection of these alterations, however a particular attention is needed for the interpretation of the expression imbalance.

Pancreatic cancer is one of the most aggressive diseases with a very poor outcome. Olaparib, a PARP inhibitor, as maintenance therapy showed benefits in patients with metastatic pancreatic adenocarcinoma bearing germline BRCA1/2 mutations. However, germline BRCA mutation has been described in only 4–7% of patients with pancreatic adenocarcinoma. A CRISPR/Cas9-mediated system was used to knock-in the c.763G > T p.(Glu255*) and c.2133C > A p.(Cys711*) mutations in cell lines to obtain truncated BRCA1 and BRCA2 proteins, respectively. A CRISPR/Cas9 ribonucleoprotein complex was assembled for each mutation and transfected into two pancreatic cell lines (T3M4 and Capan-2) and into a breast cancer cell lines (MCF7) as control. BRCA protein levels were significantly decreased in all BRCA-depleted cells ( P < 0.05), proving the transfection efficiency of our CRISPR/Cas9 systems. As expected, the calculated olaparib IC50 were significantly reduced for all cell lines harbored BRCA1 or BRCA2 mutations compared to wild-type BRCA1 / 2 cells ( P < 0.01). Furthermore, we observed a higher induction of apoptosis after 72 h olaparib treatment in BRCA-depleted cells than in wild-type cells. This strategy might offer new insights into the management of patients with pancreatic cancer and open up new perspectives based on the in vivo use of CRISPR/Cas9 strategy.

Damage specific DNA binding protein 2 (DDB2) is originally implied in the recognition of ultraviolet-induced DNA damage and the initiation of nucleotide excision repair (NER) process. This protein also demonstrated dual roles in several cancers, acting either as an oncogene or a tumor suppressor gene depending on cancer localization. In this study, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC). The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 KO) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 ACT). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays. DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 knockout in T3M4 cells enhanced the transcription of SNAIL, ZEB1 and TWIST EMT transcription factors (EMT-TFs), increased the expression of the N-cadherin mesenchymal marker and decreased the levels of the E-cadherin epithelial marker. Conversely, DDB2 overexpression in Capan-2 cells increased E-cadherin levels and decreased N-cadherin levels. The migration and invasion properties were negatively correlated with DDB2 expression in both cell models. DDB2 has no effect on T3M4 cell sensitivity to chemotherapy but sensitized Capan-2 cells to 5-FU, oxaliplatin and gemcitabine. Our study highlights the potential tumor-suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target and a prognosis and predictive biomarker in patients with PDAC.