Clinical Validity of Tumor-Informed Circulating Tumor DNA Analysis in Patients Undergoing Surgery of Colorectal Metastases.

BACKGROUND: Accurate biomarkers to monitor tumor load and response in metastatic colorectal cancer patients undergoing surgery could optimize treatment regimens. OBJECTIVE: This study aimed to explore the clinical validity of tumor-informed quantification of circulating tumor DNA in blood using ultradeep sequencing. DESIGN: Resection specimens from 53 colorectal cancer patients were analyzed for tumor-specific mutations in 15 genes. These mutations were used to measure the presence of circulating tumor DNA in preoperatively collected plasma samples using hybrid capture-based sequencing. Additional postoperative measurements were performed 1 week after surgery in 16 patients. SETTINGS: The study was conducted at the Radboud University Medical Center. PATIENTS: A total of 53 colorectal cancer patients undergoing surgery of metastases were included. MAIN OUTCOME MEASURES: The detection of circulating tumor DNA. RESULTS: At least 1 tumor-specific mutation was detected in all tumor samples. In preoperative plasma samples, circulating tumor DNA was detected in 88% (37/42) of systemic treatment-naïve patients and in 55% (6/11) of patients who received preoperative chemotherapy. More specifically, circulating tumor DNA was detected in 0% (0/3) of cases with a subtotal or partial pathologic response and in 75% (6/8) of cases without a pathologic response in the resection specimen ( p = 0.06). In postoperative plasma samples, circulating tumor DNA was detected in 80% (4/5) of patients with an incomplete resection and in 0% (0/11) of those with a complete resection ( p = 0.003). LIMITATIONS: The study was limited by the heterogeneity of the cohort and the small number of postoperative plasma samples. CONCLUSIONS: These data indicate that tumor-informed circulating tumor DNA detection in the plasma of patients undergoing surgery for metastatic colorectal cancer is feasible and may have clinical value in response monitoring and predicting residual disease. Prospective studies are needed to establish the clinical utility of circulating tumor DNA analysis to guide treatment decisions in these patients. See Video Abstract at http://links.lww.com/DCR/B990 . VALIDEZ CLNICA DEL ANLISIS DE ADN DEL TUMOR CIRCULANTE INFORMADO POR EL TUMOR EN PACIENTES SOMETIDOS A CIRUGA DE METSTASIS COLORRECTALES: ANTECEDENTES:Los biomarcadores precisos para monitorear la carga tumoral y la respuesta en pacientes con cáncer colorrectal metastásico que se someten a cirugía podrían optimizar los regímenes de tratamiento.OBJETIVO:Este estudio explora la validez clínica de la cuantificación informada por el tumor del ADN tumoral circulante en sangre mediante secuenciación ultraprofunda.DISEÑO:Se analizaron muestras de resección de 53 pacientes con cáncer colorrectal en busca de mutaciones específicas del tumor en quince genes. Estas mutaciones se usaron para medir la presencia de ADN tumoral circulante en muestras de plasma recolectadas antes de la operación usando secuenciación basada en captura híbrida. Se realizaron mediciones postoperatorias adicionales una semana después de la cirugía en dieciséis pacientes.AJUSTES:El estudio se realizó en el centro médico de la universidad de Radboud.PACIENTES:Se incluyeron un total de 53 pacientes con cáncer colorrectal sometidos a cirugía de metástasis.PRINCIPALES MEDIDAS DE RESULTADO:La detección de ADN tumoral circulante.RESULTADOS:Se detectó al menos una mutación específica de tumor en todas las muestras de tumor. En muestras de plasma preoperatorias, se detectó ADN tumoral circulante en el 88% (37/42) de los pacientes sin tratamiento sistémico previo y en el 55% (6/11) de los pacientes que recibieron quimioterapia preoperatoria. Más concretamente, en el 0% (0/3) de los casos con respuesta patológica subtotal o parcial y en el 75% (6/8) de los casos sin respuesta patológica en la pieza de resección ( p = 0,06). En muestras de plasma postoperatorio se detectó ADN tumoral circulante en el 80% (4/5) de los pacientes con una resección incompleta y en el 0% (0/11) de los que tenían resección completa ( p = 0,003).LIMITACIONES:El estudio estuvo limitado por la heterogeneidad de la cohorte y el pequeño número de muestras de plasma postoperatorias.CONCLUSIONES:Estos datos indican que la detección de ADN tumoral circulante informado por el tumor en el plasma de pacientes sometidos a cirugía por cáncer colorrectal metastásico es factible y puede tener valor clínico en el control de la respuesta y la predicción de la enfermedad residual. Se necesitan estudios prospectivos para establecer la utilidad clínica del análisis de ADN tumoral circulante para guiar las decisiones de tratamiento en estos pacientes. Consulte Video Resumen en http://links.lww.com/DCR/B990 . (Traducción-Dr. Mauricio Santamaria ).

Identification of Fusion Genes and Targets for Genetically Matched Therapies in a Large Cohort of Salivary Gland Cancer Patients.

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

Clinical, Pathology, Genetic, and Molecular Features of Colorectal Tumors in Adolescents and Adults 25 Years or Younger.

BACKGROUND & AIMS: Colorectal cancers (CRCs) are rare in adolescents and adults ages 25 years or younger. We analyzed clinical, pathology, and molecular features of colorectal tumors from adolescents and young adults in an effort to improve genetic counseling, surveillance, and, ultimately, treatment and outcomes. METHODS: We analyzed clinical data and molecular and genetic features of colorectal tumor tissues from 139 adolescents or young adults (age, ≤25 y; median age, 23 y; 58% male), collected from 2000 through 2017; tumor tissues and clinical data were obtained from the nationwide network and registry of histopathology and cytopathology and The Netherlands Cancer Registry, respectively. DNA samples from tumors were analyzed for microsatellite instability, mutations in 56 genes, and genome-wide somatic copy number aberrations. RESULTS: Mucinous and/or signet ring cell components were observed in 33% of tumor samples. A genetic tumor risk syndrome was confirmed for 39% of cases. Factors associated with shorter survival time included younger age at diagnosis, signet ring cell carcinoma, the absence of a genetic tumor risk syndrome, and diagnosis at an advanced stage of disease. Compared with colorectal tumors from patients ages 60 years or older in the Cancer Genome Atlas, higher proportions of tumors from adolescents or young adults were microsatellite stable with nearly diploid genomes, or contained somatic mutations in TP53 and POLE, whereas lower proportions contained mutations in APC. CONCLUSIONS: We found clinical, molecular, and genetic features of CRCs in adolescents or young adults to differ from those of patients older than age 60 years. In 39% of patients a genetic tumor risk syndrome was identified. These findings provide insight into the pathogenesis of CRC in young patients and suggest new strategies for clinical management. Performing genetic and molecular analyses for every individual diagnosed with CRC at age 25 years or younger would aid in this optimization.

Evaluation of a Hybrid Capture-Based Pan-Cancer Panel for Analysis of Treatment Stratifying Oncogenic Aberrations and Processes.

Stratification of patients for targeted and immune-based therapies requires extensive genomic profiling that enables sensitive detection of clinically relevant variants and interrogation of biomarkers, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Detection of single and multiple nucleotide variants, copy number variants, MSI, and TMB was evaluated using a commercially available next-generation sequencing panel containing 523 cancer-related genes (1.94 megabases). Analysis of formalin-fixed, paraffin-embedded tissue sections and cytologic material from 45 tumor samples showed that all previously known MSI-positive samples (n = 7), amplifications (n = 9), and pathogenic variants (n = 59) could be detected. TMB and MSI scores showed high intralaboratory and interlaboratory reproducibility (eight samples tested in 11 laboratories). For reliable TMB analysis, 20 ng DNA was shown to be sufficient, even for relatively poor-quality samples. A minimum of 20% neoplastic cells was required to minimize variations in TMB values induced by chromosomal instability or tumor heterogeneity. Subsequent analysis of 58 consecutive lung cancer samples in a diagnostic setting was successful and revealed sufficient somatic mutations to generate mutational signatures in 14 cases. In conclusion, the 523-gene assay can be applied for evaluation of multiple DNA-based biomarkers relevant for treatment selection.

Clonal Relationship Between Lichen Sclerosus, Differentiated Vulvar Intra-epithelial Neoplasia and Non HPV-related Vulvar Squamous Cell Carcinoma.

BACKGROUND/AIM: Differentiated vulvar intraepithelial neoplasia (dVIN) and lichen sclerosus (LS) can give rise to vulvar squamous cell carcinoma (VSCC), but genetic evidence is currently still limited. We aimed to determine genetic abnormalities in VSCC and backtrack these abnormalities in the dVIN and LS lesions. MATERIALS AND METHODS: DNA from VSCC and patient-matched dVIN and LS samples of twelve patients was collected. High-resolution genome-wide copy number analysis was performed and subsequently, we sequenced TP53. RESULTS: Copy number alterations were identified in all VSCC samples. One dVIN lesion presented with three copy number alterations that were preserved in the paired VSCC sample. Targeted sequencing of TP53 identified mutations in five VSCCs. All five mutations were traced back in the dVIN (n=5) or the LS (n=1) with frequencies ranging from 3-19%. CONCLUSION: Our data provide genetic evidence for a clonal relationship between VSCC and dVIN or LS.

Two types of primary mucinous ovarian tumors can be distinguished based on their origin.

The origin of primary mucinous ovarian tumors is unknown. We explore the hypothesis that they originate from either Brenner tumors or teratomas and examine differences between the tumors that arise in these settings. A total of 104 Brenner tumor-associated mucinous tumors and 58 teratoma-associated mucinous tumors were analyzed. Immunohistochemistry for 21 antigens and fluorescence in situ hybridization for ERBB2 and MYC were performed. Genome-wide copy number analysis and mutation analysis for 56 cancer-related genes was carried out on a subset of mucinous ovarian tumors and their complementary Brenner tumor or teratoma. Patients with teratoma-associated mucinous tumors were significantly younger than patients with Brenner tumor-associated mucinous tumors (43 vs. 61 years). During progression from cystadenoma to atypical proliferative mucinous (borderline) tumor to carcinoma expression of typical gastrointestinal markers was increased in both Brenner tumor-associated and teratoma-associated mucinous tumors. Brenner tumor-associated mucinous tumors showed more frequently calcifications and Walthard cell nests, rarely expressed SATB2 and showed more often co-deletion of CDKN2A and MTAP. Teratoma-associated mucinous tumors were characterized by mucinous stromal dissection, SATB2 expression and RNF43 mutations. Other frequent mutations in both Brenner tumor-associated and teratoma-associated mucinous tumors were TP53 and KRAS mutations. Based on identical mutations or copy number profiles clonal relationships were indicated in two mucinous tumors and their associated Brenner tumor. Teratomas and Brenner tumors give rise to different subtypes of mucinous ovarian tumors. Subsequent progression pathways are comparable since both Brenner tumor-associated and teratoma-associated mucinous tumors develop a gastrointestinal immunophenotype during progression and show early mutations in KRAS and TP53. Teratoma-associated mucinous tumors may more closely resemble true gastrointestinal tumors, indicated by their expression of SATB2 and the presence of RNF43 mutations.

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.

Comprehensive molecular and clinicopathological analysis of vascular malformations: A study of 319 cases.

Vascular malformations are part of overgrowth syndromes characterized by somatic mosaic mutations or rarely by germline mutations. Due to their similarities and diversity, clinicopathological classification can be challenging. A comprehensive targeted Next Generation Sequencing screen using Unique Molecular Identifiers with a technical sensitivity of 1% mutant alleles was performed for frequently mutated positions in ≥21 genes on 319 formalin-fixed paraffin-embedded samples. In 132 out of 319 cases pathogenic mosaic mutations were detected affecting genes previously linked to vascular malformations e.g. PIK3CA (n=80), TEK (TIE2) (n=11), AKT1 (n=1), GNAQ (n=7), GNA11 (n=4), IDH1 (n=3), KRAS (n=9), and NRAS (n=1). Six cases harbored a combination of mutations in PIK3CA and in GNA11 (n=2), GNAQ (n=2), or IDH1 (n=2). Aberrations in PTEN and RASA1 with a variant allele frequency approaching 50% suggestive of germline origin were identified in six out of 102 cases tested; four contained a potential second hit at a lower allele frequency. Ninety-one of the total 142 pathogenic mutations were present at a variant allele frequency <10% illustrating the importance of sensitive molecular analysis. Clinicopathological characteristics showed a broad spectrum and overlap when correlated with molecular data. Sensitive screening of recurrently mutated genes in vascular malformations may help to confirm the diagnosis and reveals potential therapeutic options with a significant contribution of PIK3CA/mTOR and RAS-MAPK pathway mutations. The co-existence of two activating pathogenic mutations in parallel pathways illustrates potential treatment challenges and underlines the importance of multigene testing. Detected germline mutations have major clinical impact.

Clinical and Molecular Characteristics May Alter Treatment Strategies of Thyroid Malignancies in DICER1 Syndrome.

Context: DICER1 syndrome is a rare autosomal-dominantly inherited disorder that predisposes to a variety of cancerous and noncancerous tumors of mostly pediatric and adolescent onset, including differentiated thyroid carcinoma (DTC). DTC has been hypothesized to arise secondarily to the increased prevalence of thyroid hyperplastic nodules in syndromic patients. Objective: To determine somatic alterations in DICER1-associated DTC and to study patient outcomes. Design: Retrospective series. Setting: Tertiary referral centers. Patients: Ten patients with germline pathogenic DICER1 variants and early-onset DTC. Methods: Somatic DICER1 mutation analysis, extensive somatic DNA variant and gene fusion analyses were performed on all tumors. Results: Median age at DTC diagnosis was 13.5 years and there was no recurrent or metastatic disease (median follow-up, 8 years). All thyroid specimens showed diffuse nodular hyperplasia with at least one focus suspicious of DTC but without infiltrative growth, extrathyroidal extension, vascular invasion, or lymph node metastasis. Most of the individual nodules (benign and malignant) sampled from the 10 tumors harbored distinct DICER1 RNase IIIb hotspot mutations, indicating a polyclonal composition of each tumor. Furthermore, nine of 10 DICER1-related DTCs lacked well-known oncogenic driver DNA variants and gene rearrangements. Conclusion: On the basis of our clinical, histological, and molecular data, we consider that most DICER1-related DTCs form a low-risk subgroup. These tumors may arise within one of multiple benign monoclonal nodules; thus, hemi-thyroidectomy or, more likely, total thyroidectomy may often be required. However, radioiodine treatment may be unnecessary given the patients' ages and the tumors' low propensity for metastases.

Role of germline aberrations affecting CTNNA1, MAP3K6 and MYD88 in gastric cancer susceptibility.

BACKGROUND: In approximately 10% of all gastric cancer (GC) cases, a heritable cause is suspected. A subset of these cases have a causative germline CDH1 mutation; however, in most cases the cause remains unknown. Our objective was to assess to what extent these remaining cases may be explained by germline mutations in the novel candidate GC predisposing genes CTNNA1, MAP3K6 or MYD88. METHODS: We sequenced a large cohort of unexplained young and/or familial patients with GC (n=286) without a CDH1germline mutation for germline variants affecting CTNNA1, MAP3K6 and MYD88 using a targeted next-generation sequencing approach based on single-molecule molecular inversion probes. RESULTS: Predicted deleterious germline variants were not encountered in MYD88, but recurrently observed in CTNNA1 (n=2) and MAP3K6 (n=3) in our cohort of patients with GC. In contrast to deleterious variants in CTNNA1, deleterious variants in MAP3K6 also occur frequently in the general population. CONCLUSIONS: Based on our results MAP3K6 should no longer be considered a GC predisposition gene, whereas deleterious CTNNA1 variants are confirmed as an infrequent cause of GC susceptibility. Biallelic MYD88 germline mutations are at most a very rare cause of GC susceptibility as no additional cases were identified.

High Yield of Pathogenic Germline Mutations Causative or Likely Causative of the Cancer Phenotype in Selected Children with Cancer.

Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer.Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: (1) intellectual disability and/or congenital anomalies, (2) multiple malignancies, (3) family history of cancer, or (4) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome.Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 (n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy (EP300-based Rubinstein-Taybi syndrome, ARID1A-based Coffin-Siris syndrome, ACTB-based Baraitser-Winter syndrome, and EZH2-based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2, which are possibly involved in genetic cancer predisposition.Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594-603. ©2018 AACR.

A molecular inversion probe-based next-generation sequencing panel to detect germline mutations in Chinese early-onset colorectal cancer patients.

The currently known Mendelian colorectal cancer (CRC) predisposition syndromes account for ~5-10% of all CRC cases, and are caused by inherited germline mutations in single CRC predisposing genes. Using molecular inversion probes (MIPs), we designed a targeted next-generation sequencing panel to identify mutations in seven CRC predisposing genes: APC, MLH1, MSH2, MSH6, PMS2, MUTYH and NTHL1. From a consecutive series of 2,371 Chinese CRC patients, 140 familial and non-familial cases were selected that were diagnosed with CRC at or below the age of 35 years. Through MIP-based sequencing we identified pathogenic variants in six genes in 16 out of the 140 (11.4%) patients selected. In 10 patients, known pathogenic mutations in APC (five patients), MLH1 (three patients), or MSH2 (two patients) were identified. Three additional patients were found to carry novel, likely pathogenic truncating (n = 2) and missense (n = 1) mutations in the MSH2 gene and a concomitant loss of expression of both the MSH2 and MSH6 proteins in their respective tumor tissues. From our data, we conclude that targeted MIP-based sequencing is a reliable and cost-efficient approach to identify patients with a Mendelian CRC syndrome.

Reliable Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue Using Single Molecule Tags.

Sequencing of tumor DNA to detect genetic aberrations is becoming increasingly important, not only to refine cancer diagnoses but also to predict response to targeted treatments. Next-generation sequencing is widely adopted in diagnostics for the analyses of DNA extracted from routinely processed formalin-fixed, paraffin-embedded tissue, fine-needle aspirates, or cytologic smears. PCR-based enrichment strategies are usually required to obtain sufficient read depth for reliable detection of genetic aberrations. However, although the read depth relates to sensitivity and specificity, PCR duplicates generated during target enrichment may result in overestimation of library complexity, which may result in false-negative results. Here, we report the validation of a 23-gene panel covering 41 hotspot regions using single-molecule tagging of DNA molecules by single-molecule molecular inversion probes (smMIPs), allowing assessment of library complexity. The smMIP approach outperforms Sanger and Ampliseq-Personal Genome Machine-based sequencing in our clinical diagnostic setting. Furthermore, single-molecule tags allow consensus sequence read formation, allowing detection to 1% allele frequency and reliable exclusion of variants to 3%. The number of false-positive calls is also markedly reduced (>10-fold), and our panel design allows for distinction between true mutations and deamination artifacts. Not only is this technique superior, smMIP-based library preparation is also scalable, easy to automate, and flexible. We have thus implemented this approach for sequence analysis of clinical samples in our routine diagnostic workflow.

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.

Microarray-based genomic profiling and in situ hybridization on fibrotic bone marrow biopsies for the identification of numerical chromosomal abnormalities in myelodysplastic syndrome.

BACKGROUND: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological malignancies. In MDS patients with a fibrotic bone marrow the aspiration of cells often fails (dry-tap), which hampers standard karyotyping. Obtaining genetic data from these fibrotic marrows is therefore challenging, and up till now in situ hybridization applied to bone marrow biopsies is the only option. The microarray-based genomic profiling technology has already proven its value for bone marrow aspirates and peripheral blood samples, but has never been applied to the technically challenging bone marrow biopsies. We describe an approach for microarray-based genomic profiling on bone marrow biopsies and demonstrate its ability to obtain clinically relevant cytogenetic aberrations. In addition the data were compared with those obtained by in situ hybridization and karyotyping. RESULTS: We have evaluated the success rate of microarray-based genomic profiling by studying twenty-one bone marrow biopsies (7 fibrotic MDS, 12 non-fibrotic MDS and 2 reactive), by microarray-based genomic profiling and in situ hybridization (12 of 21 cases). The data obtained with these techniques were compared with conventional karyotyping data on corresponding bone marrow aspirates. Of the 15 copy number aberrations that were detected by in situ hybridization, 13 were concordant with microarray-based genomic profiling and karyotyping, whereas two hybridizations were misinterpreted. In 20 of 21 patients, the data obtained by microarray-based genomic profiling and karyotyping were identical or differences could be explained by the presence of marker chromosomes, complex karyotypes, clonal heterogeneity or disease progression. CONCLUSIONS: We demonstrate that genome wide microarray-based genomic profiling performed on bone marrow biopsies has a similar success rate compared to in situ hybridization, and prevents misinterpretation of chromosomal losses as observed by FISH. In addition, equal to even higher resolutions were obtained with genomic profiling compared to conventional karyotyping. Our findings indicate that microarray-based profiling, even on bone marrow biopsies, is a valid approach for the identification of genetic abnormalities. This is a valuable substitution in cases of fibrotic MDS lacking cytogenetic results.

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.

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.

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.

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.