Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse.

INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Upfront Treatment Influences the Composition of Genetic Alterations in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p < 0.001) in ALL8 and ALL9 relapses compared to diagnosis, respectively, whereas no significant enrichment was found for relapses that were observed after treatment with ALL10. Furthermore, IKZF1 deletions were more frequently preserved from a major clone at diagnosis in relapses after ALL9 compared to relapses after ALL8 and ALL10 (p = 0.03). These data are in line with previous studies showing that the prognostic value of IKZF1 deletions differs between upfront protocols and is particularly strong in the ALL9 regimen. In conclusion, our data reveal a correlation between upfront treatment and the genetic composition of relapsed BCP-ALL.

Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

NTHL1 and MUTYH polyposis syndromes: two sides of the same coin?

It is now well established that germline genomic aberrations can underlie high-penetrant familial polyposis and colorectal cancer syndromes, but a genetic cause has not yet been found for the major proportion of patients with polyposis. Since next-generation sequencing has become widely accessible, several novel, but rare, high-penetrant risk factors for adenomatous polyposis have been identified, all operating in pathways responsible for genomic maintenance and DNA repair. One of these is the base excision repair pathway. In addition to the well-established role of the DNA glycosylase gene MUTYH, biallelic mutations in which predispose to MUTYH-associated polyposis, a second DNA glycosylase gene, NTHL1, has recently been associated with adenomatous polyposis and a high colorectal cancer risk. Both recessive polyposis syndromes are associated with increased risks for several other cancer types as well, but the spectrum of benign and malignant tumours in individuals with biallelic NTHL1 mutations was shown to be broader; hence the name NTHL1-associated tumour syndrome. Colorectal tumours encountered in patients with these syndromes show unique, clearly distinct mutational signatures that may facilitate the identification of these syndromes. On the basis of the prevalence of pathogenic MUTYH and NTHL1 variants in the normal population, we estimate that the frequency of the novel NTHL1-associated tumour syndrome is five times lower than that of MUTYH-associated polyposis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of Novel Candidate Genes for Early-Onset Colorectal Cancer Susceptibility.

Approximately 25-30% of colorectal cancer (CRC) cases are expected to result from a genetic predisposition, but in only 5-10% of these cases highly penetrant germline mutations are found. The remaining CRC heritability is still unexplained, and may be caused by a hitherto-undefined set of rare variants with a moderately penetrant risk. Here we aimed to identify novel risk factors for early-onset CRC using whole-exome sequencing, which was performed on a cohort of CRC individuals (n = 55) with a disease onset before 45 years of age. We searched for genes that were recurrently affected by rare variants (minor allele frequency ≤ 0.001) with potentially damaging effects and, subsequently, re-sequenced the candidate genes in a replication cohort of 174 early-onset or familial CRC individuals. Two functionally relevant genes with low frequency variants with potentially damaging effects, PTPN12 and LRP6, were found in at least three individuals. The protein tyrosine phosphatase PTP-PEST, encoded by PTPN12, is a regulator of cell motility and LRP6 is a component of the WNT-FZD-LRP5-LRP6 complex that triggers WNT signaling. All variants in LRP6 were identified in individuals with an extremely early-onset of the disease (≤30 years of age), and two of the three variants showed increased WNT signaling activity in vitro. In conclusion, we present PTPN12 and LRP6 as novel candidates contributing to the heterogeneous susceptibility to CRC.

Next generation sequencing in synovial sarcoma reveals novel gene mutations.

Over 95% of all synovial sarcomas (SS) share a unique translocation, t(X;18), however, they show heterogeneous clinical behavior. We analyzed multiple SS to reveal additional genetic alterations besides the translocation. Twenty-six SS from 22 patients were sequenced for 409 cancer-related genes using the Comprehensive Cancer Panel (Life Technologies, USA) on an Ion Torrent platform. The detected variants were verified by Sanger sequencing and compared to matched normal DNAs. Copy number variation was assessed in six tumors using the Oncoscan array (Affymetrix, USA). In total, eight somatic mutations were detected in eight samples. These mutations have not been reported previously in SS. Two of these, in KRAS and CCND1, represent known oncogenic mutations in other malignancies. Additional mutations were detected in RNF213, SEPT9, KDR, CSMD3, MLH1 and ERBB4. DNA alterations occurred more often in adult tumors. A distinctive loss of 6q was found in a metastatic lesion progressing under pazopanib, but not in the responding lesion. Our results emphasize t(X;18) as a single initiating event in SS and as the main oncogenic driver. Our results also show the occurrence of additional genetic events, mutations or chromosomal aberrations, occurring more frequently in SS with an onset in adults.

A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer.

The genetic cause underlying the development of multiple colonic adenomas, the premalignant precursors of colorectal cancer (CRC), frequently remains unresolved in patients with adenomatous polyposis. Here we applied whole-exome sequencing to 51 individuals with multiple colonic adenomas from 48 families. In seven affected individuals from three unrelated families, we identified a homozygous germline nonsense mutation in the base-excision repair (BER) gene NTHL1. This mutation was exclusively found in a heterozygous state in controls (minor allele frequency of 0.0036; n = 2,329). All three families showed recessive inheritance of the adenomatous polyposis phenotype and progression to CRC in at least one member. All three affected women developed an endometrial malignancy or premalignancy. Genetic analysis of three carcinomas and five adenomas from different affected individuals showed a non-hypermutated profile enriched for cytosine-to-thymine transitions. We conclude that a homozygous loss-of-function germline mutation in the NTHL1 gene predisposes to a new subtype of BER-associated adenomatous polyposis and CRC.

Candidate colorectal cancer predisposing gene variants in Chinese early-onset and familial cases.

AIM: To investigate whether whole-exome sequencing may serve as an efficient method to identify known or novel colorectal cancer (CRC) predisposing genes in early-onset or familial CRC cases. METHODS: We performed whole-exome sequencing in 23 Chinese patients from 21 families with non-polyposis CRC diagnosed at ≤ 40 years of age, or from multiple affected CRC families with at least 1 first-degree relative diagnosed with CRC at ≤ 55 years of age. Genomic DNA from blood was enriched for exome sequences using the SureSelect Human All Exon Kit, version 2 (Agilent Technologies) and sequencing was performed on an Illumina HiSeq 2000 platform. Data were processed through an analytical pipeline to search for rare germline variants in known or novel CRC predisposing genes. RESULTS: In total, 32 germline variants in 23 genes were identified and confirmed by Sanger sequencing. In 6 of the 21 families (29%), we identified 7 mutations in 3 known CRC predisposing genes including MLH1 (5 patients), MSH2 (1 patient), and MUTYH (biallelic, 1 patient), five of which were reported as pathogenic. In the remaining 15 families, we identified 20 rare and novel potentially deleterious variants in 19 genes, six of which were truncating mutations. One previously unreported variant identified in a conserved region of EIF2AK4 (p.Glu738_Asp739insArgArg) was found to represent a local Chinese variant, which was significantly enriched in our early-onset CRC patient cohort compared to a control cohort of 100 healthy Chinese individuals scored negative by colonoscopy (33.3% vs 7%, P < 0.001). CONCLUSION: Whole-exome sequencing of early-onset or familial CRC cases serves as an efficient method to identify known and potential pathogenic variants in established and novel candidate CRC predisposing genes.

Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development.

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.

The cancer genome: from structure to function.

The 2014 joint meeting of the International Society for Cellular Oncology (ISCO) and the European Workshop on Cytogenetics and Molecular Genetics of Solid Tumors (EWCMST), organized by Nick Gilbert, Juan Cigudosa and Bauke Ylstra, was held from 11 to 14 May in Malaga, Spain. Since the previous meeting in 2012, the ever increasing availability of new sequencing technologies has enabled the analysis of cancer genomes at an increasingly greater detail. In addition to structural changes in the genome (i.e., translocations, deletions, amplifications), frequent mutations in important regulatory genes have been found to occur, as also frequent alterations in a large number of epigenetic factors. The challenge now is to relate structural changes in cancer genomes to the underlying disease mechanisms and to reveal opportunities for the design of novel (targeted) therapies. During the meeting, various topics related to these challenges and opportunities were addressed, including those dealing with functional genomics, genome instability, biomarkers and diagnostics, cancer genetics and epigenomics. Special attention was paid to therapy-driven cancer evolution (keynote lecture) and relationships between DNA repair, cancer and ageing (Prof. Ploem lecture). Based on the information presented at the meeting, several aspects of the cancer genome and its functional implications are provided in this report.

Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.

BACKGROUND: Characteristic genomic abnormalities in patients with B cell chronic lymphocytic leukemia (CLL) have been shown to provide important prognostic information. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA), currently used in clinical diagnostics of CLL, are targeted tests aimed at specific genomic loci. Microarray-based genomic profiling is a new high-resolution tool that enables genome-wide analyses. The aim of this study was to compare two recently launched genomic microarray platforms, i.e., the CytoScan HD Array (Affymetrix) and the HumanOmniExpress Array (Illumina), with FISH and MLPA to ascertain whether these latter tests can be replaced by either one of the microarray platforms in a clinical diagnostic setting. RESULT: Microarray-based genomic profiling and FISH were performed in all 28 CLL patients. For an unbiased comparison of the performance of both microarray platforms 9 patients were evaluated on both platforms, resulting in the identification of exactly identical genomic aberrations. To evaluate the detection limit of the microarray platforms we included 7 patients in which the genomic abnormalities were present in a relatively low percentage of the cells (range 5-28%) as previously determined by FISH. We found that both microarray platforms allowed the detection of copy number abnormalities present in as few as 16% of the cells. In addition, we found that microarray-based genomic profiling allowed the identification of genomic abnormalities that could not be detected by FISH and/or MLPA, including a focal TP53 loss and copy neutral losses of heterozygosity of chromosome 17p. CONCLUSION: From our results we conclude that although the microarray platforms exhibit a somewhat lower limit of detection compared to FISH, they still allow the detection of copy number abnormalities present in as few as 16% of the cells. By applying similar interpretation criteria, the results obtained from both platforms were comparable. In addition, we conclude that both microarray platforms allow the identification of additional potential prognostic relevant abnormalities such as focal TP53 deletions and copy neutral losses of heterozygosity of chromosome 17p, which would have remained undetected by FISH or MLPA. The prognostic relevance of these novel genomic alterations requires further evaluation in prospective clinical trials.

Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24h after venipuncture.

OBJECTIVES: Circulating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS). DESIGN AND METHODS: Whole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6h, 24 and 48h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads. RESULTS: Both T21 samples were correctly identified as such at all time-points. However, after 48h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48h. CONCLUSIONS: The quality and quantity of ccffDNA extracted from plasma samples processed up to 24h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction.

Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are risk factors for colorectal cancer.

The spindle assembly checkpoint controls proper chromosome segregation during mitosis and prevents aneuploidy-an important feature of cancer cells. We performed genome-wide and targeted copy number and mutation analyses of germline DNA from 208 patients with familial or early-onset (40 years of age or younger) colorectal cancer; we identified haploinsufficiency or heterozygous mutations in the spindle assembly checkpoint genes BUB1 and BUB3 in 2.9% of them. Besides colorectal cancer, these patients had variegated aneuploidies in multiple tissues and variable dysmorphic features. These results indicate that mutations in BUB1 and BUB3 cause mosaic variegated aneuploidy and increase the risk of colorectal cancer at a young age.

EPCAM deletion carriers constitute a unique subgroup of Lynch syndrome patients.

Lynch syndrome, one of the most common cancer susceptibility syndromes, is caused by germline mutations of genes affecting the mismatch repair proteins MLH1, MSH2, MSH6 or PMS2. Most of these mutations disrupt the open reading frame of the genes involved and, as such, lead to constitutive inactivation of the mutated allele. In a subset of Lynch syndrome patients MSH2 was found to be specifically inactivated in cell lineages exhibiting EPCAM expression. These patients carry deletions of the 3' end of the EPCAM gene, including its polyadenylation signal. Due to concomitant transcriptional read-through of EPCAM, the promoter of MSH2 15 kb further downstream becomes inactivated through hypermethylation. As these 3' EPCAM deletions occur in the germline, this MSH2 promoter methylation ('epimutation') is heritable. Worldwide, numerous EPCAM 3' end deletions that differ in size and location have been detected. The risk of colorectal cancer in carriers of such EPCAM deletions is comparable to that of MSH2 mutation carriers, and is in accordance with a high expression of EPCAM in colorectal cancer stem cells. The risk of endometrial cancer in the entire group of EPCAM deletion carriers is significantly lower than that in MSH2 mutation carriers, but the actual risk appears to be dependent on the size and location of the EPCAM deletion. These observations may have important implications for the surveillance of EPCAM deletion carriers and, thus, calls for an in-depth assessment of clinically relevant genotype-phenotype correlations and its underlying molecular mechanism(s).

New insights and clinical advancements in cellular oncology.

The first joint meeting of the International Society for Cellular Oncology (ISCO) and the European Workshop on Cytogenetics and Molecular Genetics of Solid Tumors (EWCMST), organized by Bauke Ylstra, Juan Cigudosa and Nick Gilbert, was held from 4 to 8 March, 2012 in Palma de Mallorca, Spain. This meeting provided a novel and unique opportunity to jointly present the latest updates on the genetics of cancer and its implications for diagnosis, prognosis and therapy, now and in the future. Various aspects were highlighted, including the identification of effective therapeutic targets, the role of cellular senescence in tumor development and therapy, chromosome translocations in leukemias and solid tumors, mechanisms underlying fragile sites and chromosome instability, tumor-associated 'omics' landscapes, genetic and epidemiologic risk factors, the role of tissue and cancer stem cells, angiogenesis and the tumor micro-environment, and the epigenetics of cancer. In this report, new insights and clinical advancements related to these various topics are provided, based on information presented at the meeting.

Non-invasive prenatal diagnosis of fetal aneuploidies using massively parallel sequencing-by-ligation and evidence that cell-free fetal DNA in the maternal plasma originates from cytotrophoblastic cells.

Blood plasma of pregnant women contains circulating cell-free fetal DNA (ccffDNA), originating from the placenta. The use of this DNA for non-invasive detection of fetal aneuploidies using massively parallel sequencing (MPS)-by-synthesis has been proven previously. Sequence performance may, however, depend on the MPS platform and therefore we have explored the possibility for multiplex MPS-by-ligation, using the Applied Biosystems SOLiD(™) 4 system. DNA isolated from plasma samples from 52 pregnant women, carrying normal or aneuploid fetuses, was sequenced in multiplex runs of 4, 8 or 16 samples simultaneously. The sequence reads were mapped to the human reference genome and quantified according to their genomic location. In case of a fetal aneuploidy, the number of reads of the aberrant chromosome is expected to be higher or lower than in normal reference samples. To statistically determine this, Z-scores per chromosome were calculated as described previously, with thresholds for aneuploidies set at > +3.0 and < -3.0 for chromosomal over- or underrepresentation, respectively. All samples from fetal aneuploidies yielded Z-scores outside the thresholds for the aberrant chromosomes, with no false negative or positive results. Full-blown fetal aneuploidies can thus be reliably detected in maternal plasma using a multiplex MPS-by-ligation approach. Furthermore, the results obtained with a sample from a pregnancy with 45,X in the cytotrophoblastic cell layer and 46,XX in the mesenchymal core cells show that ccffDNA originates from the cytotrophoblastic cell layer. Discrepancies between the genetic constitution of this cell layer and the fetus itself are well known, and therefore, care should be taken when translating results to the fetus itself.

Revertant somatic mosaicism by mitotic recombination in dyskeratosis congenita.

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.

The epigenetics of (hereditary) colorectal cancer.

In the last decade, it has become apparent that not only DNA sequence variations but also epigenetic modifications may contribute to disease, including cancer. These epigenetic modifications involve histone modification including acetylation and methylation, DNA methylation, and chromatin remodeling. One of the best-characterized epigenetic changes is aberrant methylation of cytosines that occur in so-called CpG islands. DNA hypomethylation, prevalent as a genome-wide event, usually occurs in more advanced stages of tumor development. In contrast, DNA hypermethylation is often observed as a discrete, targeted event within tumor cells, resulting in specific loss of gene expression. Interestingly, it was found that sporadic and inherited cancers may exhibit similar DNA methylation patterns, and many genes that are mutated in familial cancers have also been found to be hypermethylated, mutated, or deleted in sporadic cancers. In this review, we will focus on DNA methylation events as heritable epimutations predisposing to colorectal cancer development.