Plasma ctDNA Monitoring of a PTCH1-Mutant Metastatic Adult Medulloblastoma Showing a Durable Benefit With Vismodegib.

Adult medulloblastoma (MB) is a rare disease affecting 0.6 persons per million adults over 19 years of age. The SHH-activated/TP53-wild type is the most common subtype, accounting for 60% of adult MBs, being characterized by mutations in PTCH1, SMO, or the TERT promoter. Several small studies demonstrate objective but short-lived responses to SMO inhibitors such as vismodegib or sonidegib. Like other oncogene-addicted solid tumors, detection of the corresponding drivers through liquid biopsy could aid in the molecular diagnosis and monitoring of the disease through less invasive procedures. However, most studies have only evaluated cerebrospinal fluid as the ctDNA reservoir, and very limited evidence exists on the role of liquid biopsy in plasma in patients with primary central nervous system tumors, including MB. We present the case of a 26-year-old patient with a recurrent MB, in which next-generation sequencing (FoundationOne CDx) revealed a mutation in PTCH1, allowing the patient to be treated with vismodegib in second line, resulting in a durable benefit lasting for 1 year. Using an in-house digital PCR probe, the PTCH1 mutation could be tracked in ctDNA during treatment with first-line chemotherapy and while on treatment with vismodegib, demonstrating a precise correlation with the radiological and clinical behavior of the disease.

Real-World Use of Highly Sensitive Liquid Biopsy Monitoring in Metastatic Breast Cancer Patients Treated with Endocrine Agents after Exposure to Aromatase Inhibitors.

Endocrine-resistant, hormone receptor-positive, and HER2-negative (HR+/HER2-) metastatic breast cancer (mBC) is largely governed by acquired mutations in the estrogen receptor, which promote ligand-independent activation, and by truncal alterations in the PI3K signaling pathway, with a broader range of gene alterations occurring with less prevalence. Circulating tumor DNA (ctDNA)-based technologies are progressively permeating the clinical setting. However, their utility for serial monitoring has been hindered by their significant costs, inter-technique variability, and real-world patient heterogeneity. We interrogated a longitudinal collection of 180 plasma samples from 75 HR+/HER2- mBC patients who progressed or relapsed after exposure to aromatase inhibitors and were subsequently treated with endocrine therapy (ET) by means of highly sensitive and affordable digital PCR and SafeSEQ sequencing. Baseline PIK3CA and TP53 mutations were prognostic of a shorter progression-free survival in our population. Mutant PIK3CA was prognostic in the subset of patients receiving fulvestrant monotherapy after progression to a CDK4/6 inhibitor (CDK4/6i)-containing regimen, and its suppression was predictive in a case of long-term benefit with alpelisib. Mutant ESR1 was prognostic in patients who did not receive concurrent CDK4/6i, an impact influenced by the variant allele frequency, and its early suppression was strongly predictive of efficacy and associated with long-term benefit in the whole cohort. Mutations in ESR1, TP53, and KRAS emerged as putative drivers of acquired resistance. These findings collectively contribute to the characterization of longitudinal ctDNA in real-world cases of HR+/HER2- mBC previously exposed to aromatase inhibitors and support ongoing studies either targeting actionable alterations or leveraging the ultra-sensitive tracking of ctDNA.

Clinical Behavior, Mutational Profile and T-Cell Repertoire of High-Grade Neuroendocrine Tumors of the Head and Neck.

Neuroendocrine carcinomas (NECs) of the head and neck (HN) account for <1% of HN cancers (HNCs), with a 5-year overall survival (OS) <20%. This is a retrospective study of HN NECs diagnosed at our institution between 2005 and 2022. Immunohistochemistry and next-generation sequencing (NGS) were used to evaluate neuroendocrine markers, tumor mutational burden (TMB), mutational profiles and T-cell receptor repertoires. Eleven patients with high-grade HN NECs were identified (male:female ratio 6:5; median age 61 (Min-Max: 31-86)): nasoethmoidal (3), parotid gland (3), submaxillary gland (1), larynx (3) and base of tongue (1). Among n = 8 stage II/IVA/B, all received (chemo)radiotherapy with/without prior surgery or induction chemotherapy, with complete response in 7/8 (87.5%). Among n = 6 recurrent/metastatic patients, three received anti-PD1 (nivolumab (2), pembrolizumab (1)): two achieved partial responses lasting 24 and 10 months. After a median follow-up of 30 and 23.5 months since diagnosis and since recurrent/metastatic, median OS was not reached. Median TMB (n = 7) was 6.72 Mut/Mb. The most common pathogenic variants were TP53, HNF1A, SMARCB1, CDKN2A, PIK3CA, RB1 and MYC. There were 224 median TCR clones (n = 5 pts). In one patient, TCR clones increased from 59 to 1446 after nivolumab. HN NECs may achieve long-lasting survival with multimodality treatment. They harbor moderate-high TMBs and large TCR repertoires, which may explain responses to anti-PD1 agents in two patients and justify the study of immunotherapy in this disease.

Case report: Clinical success targeting BRAF-mutated, hormone receptor positive, HER2- negative advanced breast cancer patient with BRAF-inhibitor plus MEK- inhibitor.

BACKGROUND: Hormone receptor-positive, human epidermal growth factor 2-negative advanced breast cancer patients have had a wide range of therapeutical options since the incorporation of targeted therapies alongside classic chemotherapy. However, because of their disease, virtually all patients will eventually experience disease progression that might compromise their lives. Thriving investigation regarding molecular therapies has provided clinicians with new options for the treatment of many cancer patients. Dabrafenib and trametinib combination has proven useful in treating malignant melanoma patients harboring a BRAF V600E mutation, improving progression-free survival and overall survival, and it has been tested in other tumors. Here we report the case of a metastatic breast cancer patient harboring a BRAF V600E mutation that achieved complete response with dabrafenib and trametinib combination.

Genomic mapping of copy number variations influencing immune response in breast cancer.

Identification of genomic alterations that influence the immune response within the tumor microenvironment is mandatory in order to identify druggable vulnerabilities. In this article, by interrogating public genomic datasets we describe copy number variations (CNV) present in breast cancer (BC) tumors and corresponding subtypes, associated with different immune populations. We identified regulatory T-cells associated with the Basal-like subtype, and type 2 T-helper cells with HER2 positive and the luminal subtype. Using gene set enrichment analysis (GSEA) for the Type 2 T-helper cells, the most relevant processes included the ERBB2 signaling pathway and the Fibroblast Growth Factor Receptor (FGFR) signaling pathway, and for CD8+ T-cells, cellular response to growth hormone stimulus or the JAK-STAT signaling pathway. Amplification of ERBB2, GRB2, GRB7, and FGF receptor genes strongly correlated with the presence of type 2 T helper cells. Finally, only 8 genes were highly upregulated and present in the cellular membrane: MILR1, ACE, DCSTAMP, SLAMF8, CD160, IL2RA, ICAM2, and SLAMF6. In summary, we described immune populations associated with genomic alterations with different BC subtypes. We observed a clear presence of inhibitory cells, like Tregs or Th2 when specific chromosomic regions were amplified in basal-like or HER2 and luminal groups. Our data support further evaluation of specific therapeutic strategies in specific BC subtypes, like those targeting Tregs in the basal-like subtype.

Durable benefit and change in TCR clonality with nivolumab in a Lynch syndrome-associated glioma.

Germline replication-repair deficient (gRRD) gliomas are exceptional events, and only a few of them have been treated with immune checkpoint inhibitors (ICIs). Contrary to sporadic gliomas, where ICIs have failed to show any objective benefit, the very few patients with gRRD gliomas treated with ICIs to date seem to benefit from programmed-death-1 (PD-1) inhibitors, such as nivolumab or pembrolizumab, either in terms of durable responses or in terms of survival. T-cell immunohistochemistry (IHC) and T-cell receptor (TCR) repertoire using high-throughput next-generation sequencing (NGS) with the Oncomine TCR-Beta-SR assay (Thermo Fisher Scientific) were analyzed in pre- and post-nivolumab tumor biopsies obtained from a patient with a Lynch syndrome-associated glioma due to a germline pathogenic hMLH1 mutation. The aim was to describe changes in the T-cell quantity and clonality after treatment with nivolumab to better understand the role of acquired immunity in gRRD gliomas. The patient showed a slow disease progression and overall survival of 10 months since the start of anti-PD-1 therapy with excellent tolerance. A very scant T-cell infiltrate was observed both at initial diagnosis and after four cycles of nivolumab. The drastic change observed in TCR clonality in the post-nivolumab biopsy may be explained by the highly spatial and temporal heterogeneity of glioblastomas. Despite the durable benefit from nivolumab, the scant T-cell infiltrate possibly explains the lack of objective response to anti-PD-1 therapy. The major change in TCR clonality observed after nivolumab possibly reflects the evolving molecular heterogeneity in a highly pre-treated disease. An in-deep review of the available literature regarding the role of ICIs in both sporadic and gRRD gliomas was conducted.

Detection of IDH1 Mutations in Plasma Using BEAMing Technology in Patients with Gliomas.

Molecular testing using blood-based liquid biopsy approaches has not been widely investigated in patients with glioma. A prospective single-center study enrolled patients with gliomas ranging from grade II to IV. Peripheral blood (PB) was drawn at different timepoints for circulating tumour DNA (ctDNA) monitoring. Next-generation sequencing (NGS) was used for the study of isocitrate dehydrogenase 1 (IDH1) mutations in the primary tumor. Beads, Emulsion, Amplification and Magnetics (BEAMing) was used for the study of IDH1 mutations in plasma and correlated with the NGS results in the tumor. Between February 2017 and July 2018, ten patients were enrolled, six with IDH1-mutant and four with IDH1 wild-type gliomas. Among the six IDH-mutant gliomas, three had the same IDH1 mutation detected in plasma (50%), and the IDH1-positive ctDNA result was obtained in patients either at diagnosis (no treatment) or during progressive disease. While the false-negative rate reached 86% (18/21), 15 out of the 18 (83%) plasma-negative results were from PB collected from the six IDH-mutant patients at times at which there was no accompanying evidence of tumor progression, as assessed by MRI. There were no false-positive cases in plasma collected from patients with IDH1 wild-type tumors. BEAMing detected IDH1 mutations in the plasma of patients with gliomas, with a modest clinical sensitivity (true positivity rate) but with 100% clinical specificity, with complete agreement between the mutant loci detected in tumor and plasma. Larger prospective studies should be conducted to expand on these findings, and further explore the clearance of mutations in PB from IDH1-positive patients in response to therapy.

Cancer Risks Associated With BRCA1 and BRCA2 Pathogenic Variants.

PURPOSE: To provide precise age-specific risk estimates of cancers other than female breast and ovarian cancers associated with pathogenic variants (PVs) in BRCA1 and BRCA2 for effective cancer risk management. METHODS: We used data from 3,184 BRCA1 and 2,157 BRCA2 families in the Consortium of Investigators of Modifiers of BRCA1/2 to estimate age-specific relative (RR) and absolute risks for 22 first primary cancer types adjusting for family ascertainment. RESULTS: BRCA1 PVs were associated with risks of male breast (RR = 4.30; 95% CI, 1.09 to 16.96), pancreatic (RR = 2.36; 95% CI, 1.51 to 3.68), and stomach (RR = 2.17; 95% CI, 1.25 to 3.77) cancers. Associations with colorectal and gallbladder cancers were also suggested. BRCA2 PVs were associated with risks of male breast (RR = 44.0; 95% CI, 21.3 to 90.9), stomach (RR = 3.69; 95% CI, 2.40 to 5.67), pancreatic (RR = 3.34; 95% CI, 2.21 to 5.06), and prostate (RR = 2.22; 95% CI, 1.63 to 3.03) cancers. The stomach cancer RR was higher for females than males (6.89 v 2.76; P = .04). The absolute risks to age 80 years ranged from 0.4% for male breast cancer to approximately 2.5% for pancreatic cancer for BRCA1 carriers and from approximately 2.5% for pancreatic cancer to 27% for prostate cancer for BRCA2 carriers. CONCLUSION: In addition to female breast and ovarian cancers, BRCA1 and BRCA2 PVs are associated with increased risks of male breast, pancreatic, stomach, and prostate (only BRCA2 PVs) cancers, but not with the risks of other previously suggested cancers. The estimated age-specific risks will refine cancer risk management in men and women with BRCA1/2 PVs.

Transcriptomic Correlates of Immunologic Activation in Head and Neck and Cervical Cancer.

Targeting the immune system has emerged as an effective therapeutic strategy for the treatment of various tumor types, including Head and Neck Squamous Cell Carcinoma (HNSCC) and Non-small-Cell Lung Cancer (NSCLC), and checkpoint inhibitors have shown to improve patient survival in these tumor types. Unfortunately, not all cancers respond to these agents, making it necessary to identify responsive tumors. Several biomarkers of response have been described and clinically tested. As of yet what seems to be clear is that a pre-activation state of the immune system is necessary for these agents to be efficient. In this study, using established transcriptomic signatures, we identified a group of gene combination associated with favorable outcome in HNSCC linked to a higher presence of immune effector cells. CD2, CD3D, CD3E, and CXCR6 combined gene expression is associated with improved outcome of HNSCC patients and an increase of infiltrating immune effector cells. This new signature also identifies a subset of cervical squamous cell carcinoma (CSCC) patients with favorable prognosis, who show an increased presence of immune effector cells in the tumor, which outcome shows similarities with the HP-positive HNSCC cohort of patients. In addition, CD2, CD3D, CD3E, and CXCR6 signature is able to predict the best favorable prognosis in terms of overall survival of CSSC patients. Of note, these findings were not reproduced in other squamous cell carcinomas like esophageal SCC or lung SCC. Prospective confirmatory studies should be employed to validate these findings.

Genomic Mapping of Splicing-Related Genes Identify Amplifications in LSM1, CLNS1A, and ILF2 in Luminal Breast Cancer.

Alternative splicing is an essential biological process, which increases the diversity and complexity of the human transcriptome. In our study, 304 splicing pathway-related genes were evaluated in tumors from breast cancer patients (TCGA dataset). A high number of alterations were detected, including mutations and copy number alterations (CNAs), although mutations were less frequently present compared with CNAs. In the four molecular subtypes, 14 common splice genes showed high level amplification in >5% of patients. Certain genes were only amplified in specific breast cancer subtypes. Most altered genes in each molecular subtype clustered to a few chromosomal regions. In the Luminal subtype, amplifications of LSM1, CLNS1A, and ILF2 showed a strong significant association with prognosis. An even more robust association with OS and RFS was observed when expression of these three genes was combined. Inhibition of LSM1, CLNS1A, and ILF2, using siRNA in MCF7 and T47D cells, showed a decrease in cell proliferation. The mRNA expression of these genes was reduced by treatment with BET inhibitors, a family of epigenetic modulators. We map the presence of splicing-related genes in breast cancer, describing three novel genes, LSM1, CLNS1A, and ILF2, that have an oncogenic role and can be modulated with BET inhibitors.

Mapping of Genomic Vulnerabilities in the Post-Translational Ubiquitination, SUMOylation and Neddylation Machinery in Breast Cancer.

The dysregulation of post-translational modifications (PTM) transversally impacts cancer hallmarks and constitutes an appealing vulnerability for drug development. In breast cancer there is growing preclinical evidence of the role of ubiquitin and ubiquitin-like SUMO and Nedd8 peptide conjugation to the proteome in tumorigenesis and drug resistance, particularly through their interplay with estrogen receptor signaling and DNA repair. Herein we explored genomic alterations in these processes using RNA-seq and mutation data from TCGA and METABRIC datasets, and analyzed them using a bioinformatic pipeline in search of those with prognostic and predictive capability which could qualify as subjects of drug research. Amplification of UBE2T, UBE2C, and BIRC5 conferred a worse prognosis in luminal A/B and basal-like tumors, luminal A/B tumors, and luminal A tumors, respectively. Higher UBE2T expression levels were predictive of a lower rate of pathological complete response in triple negative breast cancer patients following neoadjuvant chemotherapy, whereas UBE2C and BIRC5 expression was higher in luminal A patients with tumor relapse within 5 years of endocrine therapy or chemotherapy. The transcriptomic signatures of USP9X and USP7 gene mutations also conferred worse prognosis in luminal A, HER2-enriched, and basal-like tumors, and in luminal A tumors, respectively. In conclusion, we identified and characterized the clinical value of a group of genomic alterations in ubiquitination, SUMOylation, and neddylation enzymes, with potential for drug development in breast cancer.

Safety and Efficacy of Cetuximab-Based Salvage Chemotherapy After Checkpoint Inhibitors in Head and Neck Cancer.

BACKGROUND: There are still few data on the activity and safety of cetuximab-based salvage chemotherapy after immunotherapy (SCAI) in patients with squamous cell cancer of the head and neck (SCCHN). MATERIALS AND METHODS: This was a retrospective study of patients with SCCHN who received cetuximab-based SCAI after programmed cell death protein 1 or programmed cell death ligand 1(PD[L]1) inhibitors. Overall response rate (ORR) and disease control rate (DCR) with SCAI and with last chemotherapy before immunotherapy (LCBI) by RECIST 1.1, percentage change from baseline in target lesions (PCTL), progression-free survival (PFS), overall survival (OS), treatment compliance, and toxicity were evaluated. RESULTS: Between March 2016 and November 2019, 23 patients were identified. SCAI consisted of cetuximab-based combinations (3-weekly cisplatin-5FU-cetuximab [n = 2], weekly paclitaxel-cetuximab [n = 17], weekly cisplatin-cetuximab [n = 2], weekly carboplatin-paclitaxel-cetuximab [n = 2]). ORR was 56.5% (11 partial response, 2 complete response). DCR was 78.3%. Among 13 objective responders, median best PCTL was -53.5% (range, -30% to -100%). Median OS and PFS were 12 months and 6 months, respectively. In 10 patients receiving LCBI, ORR to LCBI was 40%, whereas ORR to SCAI achieved 60%. In LCBI-treated patients, median PFS with LCBI was 8 months and median PFS and OS with SCAI were 7 months and 12 months, respectively. Reduced dose intensity of the chemotherapy and cetuximab components occurred in 82.6% and 52.2% of the patients. Grade 1 or 2 adverse events (AEs) occurred in all patients. Grade 3 or 4 AEs developed in 65%, being grade 3 in all of them except in one patient (grade 4 neutropenia). There were no treatment-related deaths. CONCLUSION: Cetuximab-based salvage chemotherapy after PD(L)1 inhibitors associated with high response rates and deep tumor reductions with a manageable safety profile. Subsequent lines of therapy may explain the long survival achieved in our series. These results invite to design studies to elucidate the best therapeutic sequence in patients with SCCHN in the immunotherapy era. IMPLICATIONS FOR PRACTICE: Cetuximab-based salvage chemotherapy (SCAI) achieved high response rates in patients with recurrent/metastatic squamous cell cancer of the head and neck (SCCHN) after progression to PD-1/PD-L1 inhibitors. Objective response rate was higher than and progression-free survival was comparable to that of chemotherapy administered before immunotherapy (IO). In most patients, SCAI consisted of weekly, well-tolerated regimens. These observations have implications for current practice because of the limited evidence to date in SCCHN and the scant therapeutic options in this disease and invite to elucidate which may be the best treatment sequence for patients with head and neck cancer in the IO era.

In silico transcriptomic mapping of integrins and immune activation in Basal-like and HER2+ breast cancer.

PURPOSE: Integrins, transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions, have been linked to several cancer-associated features. A less explored function of integrins in cancer is their role in leukocyte homing and activation. Understanding their relationship with immune cell infiltrates and immune checkpoints is an area of interest in cancer research. METHODS: The expression of 33 different integrins was evaluated in relation with breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to assess the association of the identified integrin genes with immune cell infiltration, and the TCGA and METABRIC studies to assess correlations between integrin gene expression and genomic signatures of immune activation. RESULTS: We identified 7 genes coding for integrin α and ß subunits, i.e., ITGA4, ITGB2, ITGAX, ITGB7, ITGAM, ITGAL and ITGA8, which predict a favorable prognosis in Basal-like and HER2+ breast cancers. Their expression positively correlated with the presence of immune cell infiltrates within the tumor (dendritic cells, CD4+ T-cells, neutrophils, CD8+ T-cells and B-cells), with markers of T-cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast, we found that genes coding for integrins that predicted a detrimental outcome (IBSP, ITGB3BP, ITGB6, ITGB1 and ITGAV) were not associated with any of these parameters. CONCLUSIONS: We identified an integrin signature composed of 7 genes with potential to recognize immune infiltrated and activated Basal-like and HER2+ breast cancers with a favorable prognosis.

BRIP1, a Gene Potentially Implicated in Familial Colorectal Cancer Type X.

Familial colorectal cancer Type X (FCCTX) comprises a heterogeneous group of families with an increased risk of developing colorectal cancer and other related tumors, but with mismatch repair-proficient, microsatellite-stable (MSS) tumors. Unfortunately, the genetic basis underlying their cancer predisposition remains unknown. Although pathogenic germline variants in BRIP1 increase the risk of developing hereditary ovarian cancer, the involvement of BRIP1 in hereditary colorectal cancer is still not well known. In order to identify new BRIP1 variants associated with inherited colorectal cancer, affected and nonaffected individuals from 18 FCCTX or high-risk MSS colorectal cancer families were evaluated by whole-exome sequencing, and another 62 colorectal cancer patients from FCCTX or high-risk MSS colorectal cancer families were screened by a next-generation sequencing (NGS) multigene panel. The families were recruited at the Genetic Counseling Unit of Hospital Clínico San Carlos of Madrid. A total of three different BRIP1 mutations in three unrelated families were identified. Among them, there were two frameshift variants [c.1702_1703del, p.(Asn568TrpfsTer9) and c.903del, p.(Leu301PhefsTer2)] that result in the truncation of the protein and are thus classified as pathogenic (class 5). The remaining was a missense variant [c.2220G>T, p.(Gln740His)] considered a variant of uncertain significance (class 3). The segregation and loss-of-heterozygosity studies provide evidence linking the two BRIP1 frameshift variants to colorectal cancer risk, with suggestive but not definitive evidence that the third variant may be benign. The results here presented suggest that germline BRIP1 pathogenic variants could be associated with hereditary colorectal cancer predisposition.Prevention Relevance: We suggest that BRIP1 pathogenic germline variants may have a causal role in CRC as moderate cancer susceptibility alleles and be associated with hereditary CRC predisposition. A better understanding of hereditary CRC may provide important clues to disease predisposition and could contribute to molecular diagnostics, improved risk stratification, and targeted therapeutic strategies.

A Snapshot of The Tumor Microenvironment in Colorectal Cancer: The Liquid Biopsy.

The molecular profile of liquid biopsies is emerging as an alternative to tissue biopsies in the clinical management of malignant diseases. In colorectal cancer, significant liquid biopsy-based biomarkers have demonstrated an ability to discriminate between asymptomatic cancer patients and healthy controls. Furthermore, this non-invasive approach appears to provide relevant information regarding the stratification of tumors with different prognoses and the monitoring of treatment responses. This review focuses on the tumor microenvironment components which are detected in blood samples of colorectal cancer patients and might represent potential biomarkers. Exosomes released by tumor and stromal cells play a major role in the modulation of cancer progression in the primary tumor microenvironment and in the formation of an inflammatory pre-metastatic niche. Stromal cells-derived exosomes are involved in driving mechanisms that promote tumor growth, migration, metastasis, and drug resistance, therefore representing substantial signaling mediators in the tumor-stroma interaction. Besides, recent findings of specifically packaged exosome cargo in Cancer-Associated Fibroblasts of colorectal cancer patients identify novel exosomal biomarkers with potential clinical applicability. Furthermore, additional different signals emitted from the tumor microenvironment and also detectable in the blood, such as soluble factors and non-tumoral circulating cells, arise as novel promising biomarkers for cancer diagnosis, prognosis, and treatment response prediction. The therapeutic potential of these factors is still limited, and studies are in their infancy. However, innovative strategies aiming at the inhibition of tumor progression by systemic exosome depletion, exosome-mediated circulating tumor cell capturing, and exosome-drug delivery systems are currently being studied and may provide considerable advantages in the near future.

Contribution of New Adenomatous Polyposis Predisposition Genes in an Unexplained Attenuated Spanish Cohort by Multigene Panel Testing.

Attenuated adenomatous polyposis (AAP) is a heterogeneous syndrome in terms of clinical manifestations, heritability and etiology of the disease. Genetic heterogeneity and low penetrance alleles are probably the best explanation for this variability. Certainly, it is known that APC and MUTYH are high penetrance predisposition genes for adenomatous polyposis, but they only account for 5-10% of AAP. Other new predisposition genes, such as POLE, POLD1, NTHL1, AXIN2 or MSH3, have been recently described and have been associated with AAP, but their relative contribution is still not well defined. In order to evaluate the genetic predisposition to AAP in a hospital based population, germline DNAs from 158 AAP subjects were screened for genetic variants in the coding regions and intron-exon boundaries of seven associated genes through a next-generation sequencing (NGS) custom gene panel. Splicing, segregation studies, somatic mutational screening and RNA quantitative expression assays were conducted for selected variants. In four of the probands the adenoma susceptibility could be explained by actionable mutations in APC or MUTYH, and one other patient was a double carrier of two truncating variants in both POLE and NTHL1. Furthermore, 16 additional patients harbored uncertain significance variants in the remaining tested genes. This report gives information about the contribution of the newly described adenomatous polyposis predisposition genes in a Spanish attenuated polyposis cohort. Our results highly support the convenience of NGS multigene panels for attenuated polyposis genetic screening and reveals POLE frameshift variants as a plausible susceptibility mechanism for AAP.

Circulating biomarkers for early detection and clinical management of colorectal cancer.

New non-invasive approaches that can complement and improve on current strategies for colorectal cancer (CRC) screening and management are urgently needed. A growing number of publications have documented that components of tumors, which are shed into the circulation, can be detected in the form of liquid biopsies and can be used to detect CRC at early stages, to predict response to certain therapies and to detect CRC recurrence in a minimally invasive way. The analysis of circulating tumor DNA (ctDNA), tumor-derived cells (CTC, circulating tumor cells) or circulating microRNA (miRNA) in blood and other body fluids, have a great potential to improve different aspects of CRC management. The challenge now is to find which types of components, biofluids and detection methods would be the most suitable to be applied in the different steps of CRC detection and treatment. This chapter will provide an up to date review on ctDNA, CTCs and circulating miRNAs as new biomarkers for CRC, either for clinical management or early detection, highlighting their advantages and limitations.

Comparison of the Clinical Sensitivity of the Idylla Platform and the OncoBEAM RAS CRC Assay for KRAS Mutation Detection in Liquid Biopsy Samples.

KRAS mutations are common in colorectal cancer (CRC). In this setting, mutation status determination in circulating-free DNA from blood samples (liquid biopsy) has been shown to be a viable alternative to tissue testing. The objective of this study was to compare the sensitivity of two liquid biopsy methods for detecting KRAS mutations in plasma samples from metastatic CRC patients. Samples with a positive (KRAS-MUT+) result and a mutant allelic fraction (MAF) < 5% according to the OncoBEAM RAS CRC assay were pairly analyzed by the Idylla ctKRAS Mutation Test (n = 116). In a cohort of 71 patients with at least 1 year of follow-up, the progression-free survival (PFS) was determined according to MAF values. Idylla detected KRAS mutations in 81/116 OncoBEAM KRAS-MUT+ samples with MAF < 5% and in 48/79 samples with MAF < 1%. Concordance between OncoBEAM and Idylla significantly improved at higher MAF values. PFS rates at 6 and 12 months tended to be lower in patients with MAF levels between 1% and 5% than in those with levels <1%. OncoBEAM demonstrated greater sensitivity for plasma detection of KRAS mutations than Idylla. Importantly, our data identified a "gray zone" below 1% MAF where Idylla showed reduced KRAS mutation detection, highlighting the importance of an accurate method to provide the mutational status of CRC patients.

Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report.

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.