ESMO study on the availability and accessibility of biomolecular technologies in oncology in Europe.

BACKGROUND: Access to biomolecular technologies has become an essential requirement to ensure optimal and timely treatment of patients with cancer. This study sought to provide a comprehensive overview of the availability and accessibility of biomolecular technologies to patients, the status of their use and prescription, barriers to access, and potential economic issues related to cost and reimbursement. MATERIALS AND METHODS: A total of 201 field reporters from 48 European countries submitted data through an electronic survey tool between July and December 2021. The survey methodology mirrored that from previous ESMO studies addressing the availability and accessibility of antineoplastic medicines, in Europe and worldwide. The preliminary data were posted on the ESMO website for open peer-review, and amendments were incorporated into the final report. RESULTS: Overall, basic single-gene techniques are widely available, whereas access to advanced biomolecular technologies, including large next-generation sequencing panels and complete genomic profiles, is highly heterogeneous. In most countries, advanced biomolecular technologies remain largely inaccessible in clinical practice, are limited to clinical trials or basic research, and associated with progressively increasing cost as the technique becomes more advanced. Differences also exist regarding national sequencing initiatives or molecular tumour boards. The most important barriers to multiple versus single-gene sequencing techniques are the reimbursement of the test (59% versus 24%), and the availability of a suitable medicine, either through reimbursement of treatment (48% versus 30%), off-label treatment (52% versus 35%), or clinical trial enrolment (53% versus 39%). CONCLUSIONS: Cost and availability of both treatment and test are the two main factors limiting patients' access to advanced biomolecular technologies and as a consequence to innovative anticancer strategies. In the era of precision medicine, tackling the accessibility to biomolecular technologies is a key step to reduce inequalities to transformative cancer care.

Clinical utility of circulating tumor DNA sequencing with a large panel: a National Center for Precision Medicine (PRISM) study.

BACKGROUND: Circulating tumor DNA (ctDNA) sequencing is a promising approach for tailoring therapy in patients with cancer. We report hereby the results from a prospective study where we investigated the impact of comprehensive molecular profiling of ctDNA in patients with advanced solid tumors. PATIENTS AND METHODS: Genomic analysis was performed using the FoundationOne Liquid CDx Assay [324 genes, tumor mutational burden (TMB), microsatellite instability status]. Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB). Actionable targets were classified by ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT) tier leading to molecular-based treatment suggestions wherever it was possible. RESULTS: Between December 2020 and November 2021, 1772 patients with metastatic solid tumors underwent molecular profiling. Median time to assay results was 12 days. Results were contributive for 1658 patients (94%). At least one actionable target was detected in 1059 patients (64%) with a total of 1825 actionable alterations including alteration of the DNA damage repair response pathway (n = 336, 18%), high TMB (>16 mutations/Mb; n = 243, 13%), PIK3CA mutations (n = 150, 8%), ERBB family pathway alterations (n = 127, 7%), PTEN alterations (n = 95, 5%), FGFR alterations (n = 67, 4%) and MET activations (n = 13, 0.7%). The MTB recommended a matched therapy for 597 patients (56%) with a total of 819 therapeutic orientations: clinical trials (n = 639, 78%), off-label/compassionate use (n = 81, 10%), approved drug (n = 51, 6%), and early access program (n = 48, 6%). In total, 122 patients (21%) were treated. Among the assessable patients (n = 107), 4 (4%) had complete response, 35 (33%) had partial response, 27 (25%) had stable disease, and 41 (38%) a progressive disease as best response. The median progression-free survival and median overall survival were 4.7 months (95% confidence interval 2.7-6.7 months) and 8.3 months (95% confidence interval 4.7-11.9 months) respectively. CONCLUSIONS: ctDNA sequencing with a large panel is an efficient approach to match patients with advanced cancer with targeted therapies.

The Frequency of Germline BRCA and Non-BRCA HR-Gene-Variants in a Cohort of Pancreatic Cancer Patients.

Germline DNA alterations affecting homologous recombination pathway genes have been associated with pancreatic cancer (PC) risk. BRCA2 is the most studied gene and affects the management of PC patients and their families. Even though recent reports have suggested a similar role of germline ATM pathogenic variants (PV) in familial PC, there is still a disagreement between experts on how it could affect patient management given the lack of proper PC risk estimates. We retrospectively analyzed the germline data of 257 PC patients among whom nearly 50% were sporadic cases. We showed similar frequencies of BRCA2 (4.9%) and ATM (4.4%) PV or likely pathogenic variants, which were not related to familial history. Based on our findings and that of the literature, we suggest including ATM gene among the panel of genes analyzed in PC patients pending the publication of prospective studies.

Association of location of BRCA1 and BRCA2 mutations with benefit from olaparib and bevacizumab maintenance in high-grade ovarian cancer: phase III PAOLA-1/ENGOT-ov25 trial subgroup exploratory analysis.

BACKGROUND: In the phase III PAOLA-1 study, the addition of maintenance olaparib to bevacizumab in patients with newly diagnosed high-grade ovarian cancer (HGOC) resulted in prolonged progression-free survival (PFS), particularly for homologous recombination deficiency-positive tumors, including those with a BRCA mutation (BRCAm). The magnitude of benefit from olaparib and bevacizumab according to the location of mutation in BRCA1/BRCA2 remains to be explored. PATIENTS AND METHODS: Patients with advanced-stage HGOC responding after platinum-based chemotherapy + bevacizumab received maintenance therapy bevacizumab (15 mg/kg q3w for 15 months) + either olaparib (300 mg b.i.d. for 24 months) or placebo. PFS was analyzed in the subgroup of patients with BRCA1m/BRCA2m according to mutation location in the functional domains of BRCA1 [Really Interesting Gene (RING), DNA-binding domain (DBD), or C-terminal domain of BRCA1 (BRCT)] and BRCA2 [RAD51-binding domain (RAD51-BD); DBD]. RESULTS: From 806 randomized patients, 159 harbored BRCA1m (19.7%) and 74 BRCA2m (9.2%). BRCA1m in RING, DBD, and BRCT domains was detected in 18, 40, and 33 patients, and BRCA2m in RAD51-BD and DBD in 36 and 13 patients, respectively. After a median follow-up of 25.5 months, benefit from maintenance olaparib + bevacizumab was observed irrespective of location of BRCAm. The benefit was particularly high for those with BRCA1m located in the DBD, with 24-month PFS estimated to be 89% and 15% [olaparib + bevacizumab versus placebo + bevacizumab hazard ratio = 0.08 (95% confidence interval 0.02-0.28); interaction P = 0.03]. In BRCA2m patients, 24-month PFS rates for those with mutations located in the DBD were 90% and 100% (olaparib + bevacizumab versus placebo + bevacizumab), respectively. CONCLUSIONS: Advanced-stage BRCA-mutated HGOC patients reported PFS benefit from maintenance olaparib and bevacizumab regardless of mutation location. The benefit is particularly high for patients with mutations located in the DBD of BRCA1. Mutations located in the DBD of BRCA2 are also associated with excellent outcome.

Whole exome sequencing in molecular diagnostics of cancer decreases over time: evidence from a cost analysis in the French setting.

OBJECTIVES: Although high-throughput sequencing is revolutionising medicine, data on the actual cost of whole exome sequencing (WES) applications are needed. We aimed at assessing the cost of WES at a French cancer institute in 2015 and 2018. METHODS: Actual costs of WES application in oncology research were determined using both micro-costing and gross-costing for the years 2015 and 2018, before and after the acquisition of a new sequencer. The entire workflow process of a WES test was tracked, and the number and unit price of each resource were identified at the most detailed level, from library preparation to bioinformatics analyses. In addition, we conducted an ad hoc analysis of the bioinformatics storage costs of data issued from WES analyses. RESULTS: The cost of WES has decreased substantially, from €1921 per sample (i.e. cost of €3842 per patient) in 2015 to €804 per sample (i.e. cost of €1,608 per patient) in 2018, representing a decrease of 58%. In the meantime, the cost of bioinformatics storage has increased from €19,836 to €200,711. CONCLUSION: This study suggests that WES cost has decreased significantly in recent years. WES has become affordable, even though clinical utility and efficiency still need to be confirmed.

Biomarker testing in oncology - Requirements for organizing external quality assessment programs to improve the performance of laboratory testing: revision of an expert opinion paper on behalf of IQNPath ABSL.

In personalized medicine, predictive biomarker testing is the basis for an appropriate choice of therapy for patients with cancer. An important tool for laboratories to ensure accurate results is participation in external quality assurance (EQA) programs. Several providers offer predictive EQA programs for different cancer types, test methods, and sample types. In 2013, a guideline was published on the requirements for organizing high-quality EQA programs in molecular pathology. Now, after six years, steps were taken to further harmonize these EQA programs as an initiative by IQNPath ABSL, an umbrella organization founded by various EQA providers. This revision is based on current knowledge, adds recommendations for programs developed for predictive biomarkers by in situ methodologies (immunohistochemistry and in situ hybridization), and emphasized transparency and an evidence-based approach. In addition, this updated version also has the aim to give an overview of current practices from various EQA providers.

Recommendations for the use of next-generation sequencing (NGS) for patients with metastatic cancers: a report from the ESMO Precision Medicine Working Group.

Next-generation sequencing (NGS) allows sequencing of a high number of nucleotides in a short time frame at an affordable cost. While this technology has been widely implemented, there are no recommendations from scientific societies about its use in oncology practice. The European Society for Medical Oncology (ESMO) is proposing three levels of recommendations for the use of NGS. Based on the current evidence, ESMO recommends routine use of NGS on tumour samples in advanced non-squamous non-small-cell lung cancer (NSCLC), prostate cancers, ovarian cancers and cholangiocarcinoma. In these tumours, large multigene panels could be used if they add acceptable extra cost compared with small panels. In colon cancers, NGS could be an alternative to PCR. In addition, based on the KN158 trial and considering that patients with endometrial and small-cell lung cancers should have broad access to anti-programmed cell death 1 (anti-PD1) antibodies, it is recommended to test tumour mutational burden (TMB) in cervical cancers, well- and moderately-differentiated neuroendocrine tumours, salivary cancers, thyroid cancers and vulvar cancers, as TMB-high predicted response to pembrolizumab in these cancers. Outside the indications of multigene panels, and considering that the use of large panels of genes could lead to few clinically meaningful responders, ESMO acknowledges that a patient and a doctor could decide together to order a large panel of genes, pending no extra cost for the public health care system and if the patient is informed about the low likelihood of benefit. ESMO recommends that the use of off-label drugs matched to genomics is done only if an access programme and a procedure of decision has been developed at the national or regional level. Finally, ESMO recommends that clinical research centres develop multigene sequencing as a tool to screen patients eligible for clinical trials and to accelerate drug development, and prospectively capture the data that could further inform how to optimise the use of this technology.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Breast and ovarian cancer predisposition due to de novo BRCA1 and BRCA2 mutations.

BRCA1 and BRCA2 are the two major genes predisposing to breast and ovarian cancer. Whereas high de novo mutation rates have been demonstrated for several genes, only 11 cases of de novo BRCA1/2 mutations have been reported to date and the BRCA1/2 de novo mutation rate remains unknown. The present study was designed to fill this gap based on a series of 12 805 consecutive unrelated patients diagnosed with breast and/or ovarian cancer who met the inclusion criteria for BRCA1/2 gene analysis according to French guidelines. BRCA1/2 mutations were detected in 1527 (12%) patients, and three BRCA1 mutations and one BRCA2 mutation were de novo. The BRCA1/2 de novo mutation rate was estimated to be 0.3% (0.1%; 0.7%). Although rare, it may be useful to take the possibility of de novo BRCA1/2 mutation into account in genetic counseling of relatives and to improve the understanding of complex family histories of breast and ovarian cancers.

Randomised proof-of-concept phase II trial comparing targeted therapy based on tumour molecular profiling vs conventional therapy in patients with refractory cancer: results of the feasibility part of the SHIVA trial.

BACKGROUND: The SHIVA trial is a multicentric randomised proof-of-concept phase II trial comparing molecularly targeted therapy based on tumour molecular profiling vs conventional therapy in patients with any type of refractory cancer. RESULTS of the feasibility study on the first 100 enrolled patients are presented. METHODS: Adult patients with any type of metastatic cancer who failed standard therapy were eligible for the study. The molecular profile was performed on a mandatory biopsy, and included mutations and gene copy number alteration analyses using high-throughput technologies, as well as the determination of oestrogen, progesterone, and androgen receptors by immunohistochemistry (IHC). RESULTS: Biopsy was safely performed in 95 of the first 100 included patients. Median time between the biopsy and the therapeutic decision taken during a weekly molecular biology board was 26 days. Mutations, gene copy number alterations, and IHC analyses were successful in 63 (66%), 65 (68%), and 87 (92%) patients, respectively. A druggable molecular abnormality was present in 38 patients (40%). CONCLUSIONS: The establishment of a comprehensive tumour molecular profile was safe, feasible, and compatible with clinical practice in refractory cancer patients.

KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance.

KRAS mutation testing is mandatory for patients with metastatic colorectal cancer who are eligible for treatment with an epidermal growth factor receptor targeting agent, since tumors with a mutation are not sensitive to the drug. Several methods for mutation testing are in use and the need for external quality assurance has been demonstrated. An often little addressed but important issue in external quality assurance schemes is a low percentage of tumor cells in the test samples, where the analytical sensitivity of most tests becomes critical. Using artificial samples based on a mixture of cell lines with known mutation status of the KRAS gene, we assessed the reliability of a series of commonly used methods (Sanger sequencing, high resolution melting, pyrosequencing, and amplification refractory mutation system-polymerase chain reaction) on samples with 0, 2.5, 5, 10, and 15 % mutated cells. Nine laboratories throughout Europe participated and submitted a total of ten data sets. The limit of detection of each method differed, ranging from >15-5 % tumor cells. All methods showed a decreasing correct mutation call rate proportionally with decreasing percentage of tumor cells. Our findings indicate that laboratories and clinicians need to be aware of the decrease in correct mutation call rate proportionally with decreasing percentage of tumor cells and that external quality assurance schemes need to address the issue of low tumor cell percentage in the test samples.

Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition.

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

Prenatal BACs-on-Beads™ : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

OBJECTIVE: Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes-on-Beads™ (BoBs™ ; CE-IVD), a bead-based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700. METHOD: We retrospectively studied 408 selected samples and prospectively tested 212 consecutive samples ascertained for conventional karyotyping. RESULTS: We did not find false-positive results. Triploidies were not detected. Maternal cell contamination of male samples up to 90% was unmasked inspecting gonosome profiles. Mosaic conditions at 20 to 30% were revealed. Failures were due to low amount of DNA. CONCLUSION: Prenatal BoBs™ is a robust technology for the investigation of fetuses with normal karyotype with or without sonographic abnormalities. Running in parallel with the karyotype analysis, it can be proposed instead of rapid FISH or QF-PCR providing rapid results on common aneuploidies and additional information regarding the microdeletion syndromes.

[Clinical significance of BRAF mutations in colorectal cancer]. / Intérêt clinique des mutations de BRAF dans le cancer colorectal.

BRAF mutations, present in 5 to 10% of colorectal cancers, have a proved oncogenic effect which is linked to their implication in the RAS/MAPK intracellular signalling pathway and they occurred at early stage of colorectal carcinogenesis. Many studies have therefore assessed their clinical significance as diagnostic and prognostic marker in colorectal cancers. More recently, their location downstream to EGFR and KRAS in the RAS/MAPK pathway have led to their evaluation as predictive marker of resistance to anti-EGFR monoclonal antibodies. This article aims to review the role of BRAF mutations in the diagnostic strategy of Lynch syndrome, their prognostic value in colorectal cancers and their potential value as predictive marker of resistance to anti-EGFR antibodies.

The TP53 Arg72Pro and MDM2 309G>T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93-1.10, P(trend)=0.77; MDM2: HR=0.96, 95%CI: 0.84-1.09, P(trend)=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87-1.12, P(trend)=0.83; MDM2: HR=0.98, 95%CI: 0.80-1.21, P(trend)=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.