Comprehensive Genomic Profiling Alters Clinical Diagnoses in a Significant Fraction of Tumors Suspicious of Sarcoma.

PURPOSE: Tumor classification is a key component in personalized cancer care. For soft tissue and bone tumors, this classification is currently based primarily on morphology assessment and immunohistochemical staining. However, these standard-of-care methods can pose challenges for pathologists. We therefore assessed how whole-genome and whole-transcriptome sequencing (WGTS) impacted tumor classification and clinical management when interpreted together with histomorphology. EXPERIMENTAL DESIGN: We prospectively evaluated WGTS in routine diagnostics of 200 soft tissue and bone tumors suspicious for malignancy, including DNA and RNA isolation from the tumor, and DNA isolation from a peripheral blood sample or any non-tumor tissue. RESULTS: Based on specific genomic alterations or absence of presumed findings, WGTS resulted in reclassification of 7% (13/197) of the histopathological diagnoses. Four cases were downgraded from low-grade sarcomas to benign lesions, and two cases were reclassified as metastatic malignant melanomas. Fusion genes associated with specific tumor entities were found in 30 samples. For malignant soft tissue and bone tumors, we identified treatment relevant variants in 15% of cases. Germline pathogenic variants associated to a hereditary cancer syndrome were found in 22 participants (11%). CONCLUSION: We conclude that WGTS provides an important dimension of data which aids in the classification of soft tissue and bone tumors, correcting a significant fraction of clinical diagnoses, and identifies molecular targets relevant for precision medicine. However, genetic findings need to be evaluated in their morphopathological context, just as germline findings need to be evaluated in the context of patient phenotype and family history.

Sarcoma care in the era of precision medicine.

Sarcoma subtype classification is currently mainly based upon histopathological morphology. Molecular analyses have emerged as an efficient addition to the diagnostic workup and sarcoma care. Knowledge about the sarcoma genome increases, and genetic events that can either support a histopathological diagnosis or suggest a differential diagnosis are identified, as well as novel therapeutic targets. In this review, we present diagnostic, therapeutic, and prognostic molecular markers that are, or might soon be, used clinically. For sarcoma diagnostics, there are specific fusions highly supportive or pathognomonic for a diagnostic entity-for instance, SYT::SSX in synovial sarcoma. Complex karyotypes also give diagnostic information-for example, supporting dedifferentiation rather than low-grade central osteosarcoma or well-differentiated liposarcoma when detected in combination with MDM2/CDK4 amplification. Molecular treatment predictive sarcoma markers are available for gastrointestinal stromal tumor (GIST) and locally aggressive benign mesenchymal tumors. The molecular prognostic markers for sarcomas in clinical practice are few. For solitary fibrous tumor, the type of NAB2::STAT6 fusion is associated with the outcome, and the KIT/PDGFRA pathogenic variant in GISTs can give prognostic information. With the exploding availability of sequencing technologies, it becomes increasingly important to understand the strengths and limitations of those methods and their context in sarcoma diagnostics. It is reasonable to believe that most sarcoma treatment centers will increase the use of massive-parallel sequencing soon. We conclude that the context in which the genetic findings are interpreted is of importance, and the interpretation of genomic findings requires considering tumor histomorphology.

Sensitive Detection of Cell-Free Tumour DNA Using Optimised Targeted Sequencing Can Predict Prognosis in Gastro-Oesophageal Cancer.

In this longitudinal study, cell-free tumour DNA (a liquid biopsy) from plasma was explored as a prognostic biomarker for gastro-oesophageal cancer. Both tumour-informed and tumour-agnostic approaches for plasma variant filtering were evaluated in 47 participants. This was possible through sequencing of DNA from tissue biopsies from all participants and cell-free DNA from plasma sampled before and after surgery (n = 42), as well as DNA from white blood cells (n = 21) using a custom gene panel with and without unique molecular identifiers (UMIs). A subset of the plasma samples (n = 12) was also assayed with targeted droplet digital PCR (ddPCR). In 17/31 (55%) diagnostic plasma samples, tissue-verified cancer-associated variants could be detected by the gene panel. In the tumour-agnostic approach, 26 participants (59%) had cancer-associated variants, and UMIs were necessary to filter the true variants from the technical artefacts. Additionally, clonal haematopoietic variants could be excluded using the matched white blood cells or follow-up plasma samples. ddPCR detected its targets in 10/12 (83%) and provided an ultra-sensitive method for follow-up. Detectable cancer-associated variants in plasma correlated to a shorter overall survival and shorter time to progression, with a significant correlation for the tumour-informed approaches. In summary, liquid biopsy gene panel sequencing using a tumour-agnostic approach can be applied to all patients regardless of the presence of a tissue biopsy, although this requires UMIs and the exclusion of clonal haematopoietic variants. However, if sequencing data from tumour biopsies are available, a tumour-informed approach improves the value of cell-free tumour DNA as a negative prognostic biomarker in gastro-oesophageal cancer patients.

Massive parallel sequencing in individuals with multiple primary tumours reveals the benefit of re-analysis.

Multiple primary cancers, defined as three or more primary tumours, are rare, and there are few genetic studies concerning them. There is a need for increased knowledge on the heritability of multiple primary cancers and genotype-phenotype correlations. We have performed whole-genome/exome sequencing (WGS/WES) in ten individuals with three or more primary tumours, with no previous findings on standard clinical genetic investigations. In one individual with a clinical diagnosis of MEN1, a likely pathogenic cryptic splice site variant was detected in the MEN1 gene. The variant (c.654C > A) is synonymous but we showed in a cDNA analysis that it affects splicing and leads to a frameshift, with the theoretical new amino acid sequence p.(Gly219Glufs*13). In one individual with metachronous colorectal cancers, ovarian cancer, endometrial cancer and chronic lymphocytic leukaemia, we found a likely pathogenic variant in the MLH1 gene (c.27G > A), and two risk factor variants in the genes CHEK2 and HOXB13. The MLH1 variant is synonymous but has previously been shown to be associated to constitutional low-grade hypermethylation of the MLH1 promoter, and segregates with disease in families with colorectal and endometrial cancer. No pathogenic single nucleotide or structural variants were detected in the remaining eight individuals in the study. The pathogenic variants found by WGS/WES were in genes already sequenced by Sanger sequencing and WES in the clinic, without any findings. We conclude that, in individuals with an unequivocal clinical diagnosis of a specific hereditary cancer syndrome, where standard clinical testing failed to detect a causative variant, re-analysis may lead to a diagnosis.

Massive parallel sequencing in a family with rectal cancer.

BACKGROUND: We have previously reported a family with a suspected autosomal dominant rectal and gastric cancer syndrome without any obvious causative genetic variant. Here, we focused the study on a potentially isolated rectal cancer syndrome in this family. METHODS: We included seven family members (six obligate carriers). Whole-exome sequencing and whole-genome sequencing data were analyzed and filtered for shared coding and splicing sequence and structural variants among the affected individuals. RESULTS: When considering family members with rectal cancer or advanced adenomas as affected, we found six new potentially cancer-associated variants in the genes CENPB, ZBTB20, CLINK, LRRC26, TRPM1, and NPEPL1. All variants were missense variants and none of the genes have previously been linked to inherited rectal cancer. No structural variant was found. CONCLUSION: By massive parallel sequencing in a family suspected of carrying a highly penetrant rectal cancer predisposing genetic variant, we found six genetic missense variants with a potential connection to the rectal cancer in this family. One of them could be a high-risk genetic variant, or one or more of them could be low risk variants. The p.(Glu438Lys) variant in the CENPB gene was found to be of particular interest. The CENPB protein binds DNA and helps form centromeres during mitosis. It is involved in the WNT signaling pathway, which is critical for colorectal cancer development and its role in inherited rectal cancer needs to be further examined.

Cell-free tumour DNA analysis detects copy number alterations in gastro-oesophageal cancer patients.

BACKGROUND: Analysis of cell-free tumour DNA, a liquid biopsy, is a promising biomarker for cancer. We have performed a proof-of principle study to test the applicability in the clinical setting, analysing copy number alterations (CNAs) in plasma and tumour tissue from 44 patients with gastro-oesophageal cancer. METHODS: DNA was isolated from blood plasma and a tissue sample from each patient. Array-CGH was applied to the tissue DNA. The cell-free plasma DNA was sequenced by low-coverage whole-genome sequencing using a clinical pipeline for non-invasive prenatal testing. WISECONDOR and ichorCNA, two bioinformatic tools, were used to process the output data and were compared to each other. RESULTS: Cancer-associated CNAs could be seen in 59% (26/44) of the tissue biopsies. In the plasma samples, a targeted approach analysing 61 regions of special interest in gastro-oesophageal cancer detected cancer-associated CNAs with a z-score >5 in 11 patients. Broadening the analysis to a whole-genome view, 17/44 patients (39%) had cancer-associated CNAs using WISECONDOR and 13 (30%) using ichorCNA. Of the 26 patients with tissue-verified cancer-associated CNAs, 14 (54%) had corresponding CNAs in plasma. Potentially clinically actionable amplifications overlapping the genes VEGFA, EGFR and FGFR2 were detected in the plasma from three patients. CONCLUSIONS: We conclude that low-coverage whole-genome sequencing without prior knowledge of the tumour alterations could become a useful tool for cell-free tumour DNA analysis of total CNAs in plasma from patients with gastro-oesophageal cancer.

Genetic analyses supporting colorectal, gastric, and prostate cancer syndromes.

Colorectal cancer (CRC), prostate cancer (PrC), and gastric cancer (GC) are common worldwide, and the incidence is to a certain extent dependent on genetics. We have recently shown that in families with more than one case of CRC, the risk of other malignancies is increased. We therefore suggested the presence of not yet described CRC syndromes. In this study, we have searched for genetic susceptibility loci for potential cancer syndromes involving CRC combined with PrC and/or GC. We have performed SNP (single-nucleotide polymorphism)-based linkage analyses in 45 families with CRC, PrC, and GC. In the regions with suggested linkage, we performed exome and association haplotype analyses. Five loci generated a high logarithm of odds (HLOD) score >2, suggestive of linkage, in chromosome bands 1q31-32, 1q24-25, 6q25-26, 18p11-q11, and Xp11. Exome analysis detected no potential pathogenic sequence variants. The haplotype association study showed that one of the top five haplotypes with the lowest P value in the chromosome band 6q25 interestingly was found in the family which contributed the most to the increased HLOD at that locus. This study supports a suggested hereditary cancer syndrome involving CRC and PrC and indicates a location at 6q25. The impact of this locus needs to be confirmed in additional studies.

Individuals with FANCM biallelic mutations do not develop Fanconi anemia, but show risk for breast cancer, chemotherapy toxicity and may display chromosome fragility.

PurposeMonoallelic germ-line mutations in the BRCA1/FANCS, BRCA2/FANCD1 and PALB2/FANCN genes confer high risk of breast cancer. Biallelic mutations in these genes cause Fanconi anemia (FA), characterized by malformations, bone marrow failure, chromosome fragility, and cancer predisposition (BRCA2/FANCD1 and PALB2/FANCN), or an FA-like disease presenting a phenotype similar to FA but without bone marrow failure (BRCA1/FANCS). FANCM monoallelic mutations have been reported as moderate risk factors for breast cancer, but there are no reports of any clinical phenotype observed in carriers of biallelic mutations.MethodsBreast cancer probands were subjected to mutation analysis by sequencing gene panels or testing DNA damage response genes.ResultsFive cases homozygous for FANCM loss-of-function mutations were identified. They show a heterogeneous phenotype including cancer predisposition, toxicity to chemotherapy, early menopause, and possibly chromosome fragility. Phenotype severity might correlate with mutation position in the gene.ConclusionOur data indicate that biallelic FANCM mutations do not cause classical FA, providing proof that FANCM is not a canonical FA gene. Moreover, our observations support previous findings suggesting that FANCM is a breast cancer-predisposing gene. Mutation testing of FANCM might be considered for individuals with the above-described clinical features.

Zonisamide: pharmacokinetics, efficacy, and adverse events in children with epilepsy.

BACKGROUND: Zonisamide is a new generation antiepileptic drug (AED) widely used in children with refractory epilepsy, although until recently, it was used to a large extent as off-label or unlicensed medication due to the lack of evidence-based studies. Children have a different pharmacokinetic profile than adults and an adult dose regimen cannot be directly translated into pediatric use. Patients and METHODS: In this retrospective noninterventional study of the medical records of 75 children with pharmacoresistant epilepsy, the pharmacokinetics, efficacy and safety of zonisamide were examined. The dose-to-concentration ratio, the daily weight-normalized dose of zonisamide divided by its plasma concentration, was used as a measure of clearance. In addition, data on the efficacy of zonisamide to reduce seizures and reported adverse events were extracted from the medical records and analyzed. RESULTS: Young children (range, 0-4 years) had a significantly increased zonisamide clearance compared with older ones (range, 5-17 years) and those with enzyme-inducing comedication (carbamazepine, phenobarbital, or phenytoin) had increased clearance compared with those on nonenzyme inducers; the increases were 1.7-fold and 1.8-fold, respectively. No significant difference in clearance was found between female and male subjects. The clearances of concomitant AEDs were not affected by zonisamide administration. The overall efficacy of zonisamide for reducing seizure frequency ≥ 50% was 35% and the most frequent adverse event was fatigue, reported in 23% of the patients. CONCLUSION: Patients with enzyme-inducing comedication or of young age (range, 0-4 years) might need higher weight-normalized doses to achieve the same plasma levels as in patients with no enzyme-inducing comedication or patients of older age. Zonisamide was not found to influence the pharmacokinetics of concomitant AEDs. The shortage of pharmacokinetic studies of zonisamide in children highlights the need for research of this kind.