Germline DNA Of Patients Research Articles

Comprehensive Molecular Characterization of Soft Tissue Sarcoma for Prediction of Pazopanib-Based Treatment Response.

Even though pazopanib, a multitargeted tyrosine kinase inhibitor, has been approved for refractory soft tissue sarcoma (STS), little is known about the molecular determinants of the response to pazopanib. We performed integrative molecular characterization to identify potential predictors of pazopanib efficacy. We obtained fresh pre-treatment tumor tissue from 35 patients with advanced STS receiving pazopanib-based treatment. Among those, 18 (51.4%) received pazopanib monotherapy, and the remaining 17 (48.6%) received pazopanib in combination with durvalumab, programmed death-ligand 1 blockade. Whole-exome and transcriptome sequencing were performed for each tumor and patient germline DNA. Of the 35 patients receiving pazopanib-based treatment, nine achieved a partial response (PR), resulting in an objective response rate (ORR) of 27.3%, and the median progression-free survival (PFS) was 6.0 months. Patients with CDK4 amplification (copy ratio tumor to normal > 2) exhibited shorter PFS (3.7 vs. 7.9 months, p=2.09×10-4) and a poorer response (ORR; 0% vs. 33.3%) compared to those without a gene amplification (copy ratio ≤ 2). Moreover, non-responders demonstrated transcriptional activation of CDK4 via DNA amplification, resulting in cell cycle activation. In the durvalumab combination cohort, seven of the 17 patients (41.2%) achieved a PR, and gene expression analysis revealed that durvalumab responders exhibited high immune/stromal cell infiltration, mainly comprising natural killer cells, compared to non-responders as well as increased expression of CD19, a B-cell marker. Despite the limitation of heterogeneity in the study population and treatment, we identified possible molecular predictors of pazopanib efficacy that can be employed in future clinical trials aimed at evaluating therapeutic strategies.

Utilization of Non-Invasive Sample Sources for Identification of Somatic and Germline Mutations in Hematologic Malignancies and Clonal Hematopoiesis

IntroductionA matched “tumor” and germline sample is required to unambiguously identify somatically acquired mutations in the blood or bone marrow of patients with hematologic malignancies or clonal hematopoiesis, particularly when variants of unknown significance (VUS) with a variant allele frequency (VAF) approximating 50% are encountered. Our goal was to identify sample sources for matched blood and germline samples to fit the following criteria: 1) non-invasive, 2) amenable to home collection by participants, and 3) stable at room temperature for an extended period. We hypothesized that saliva, comprised of mainly white blood cells as the DNA source, is a viable alternative to blood for identifying somatic hematopoietic mutations and that nail clippings provide adequate DNA devoid of contamination from blood cells to serve as a germline sample. To test this, a targeted NGS myeloid panel was performed on concurrently collected blood, saliva, and nail samples from patients with myeloproliferative neoplasms (MPN) or other clonal disorders.MethodsPeripheral blood was collected in EDTA tubes, aliquoted into microcentrifuge tubes, and stored at -80°C until use. Saliva was collected using a DNA/RNA shield saliva collection kit (Zymo) and stored at -80°C until use. Fingernail or toenails were clipped into a plastic bag and stored at room temperature until use. For isolation of DNA from blood the DSP DNA Blood mini kit (QIAGEN) was used. For saliva and nails, the QIAmp DNA Investigator kit (QIAGEN) was used. After DNA quantification targeted NGS libraries were prepared from 100ng of DNA per sample using the ArcherDX VariantPlex Myeloid (SK0123) workflow. The resulting libraries were sequenced on an Illumina NextSeq500 instrument using v2 chemistry (Illumina, San Diego, CA), obtaining at least 4 millionreads per sample. The FASTQ data files were analyzed on ArcherDX Suite Analysis software (v.5.1.7) to identify SNPs, Indels, structural rearrangements, and Copy Number Variations.ResultsFirst, to test if nail clippings are a feasible source of germline DNA, we performed a targeted NGS myeloid panel on paired blood and nail from a patient with Polycythemia Vera (Patient #1). Clippings from approximately five nails was sufficient to yield 100ng of DNA. JAK2 V617F was detected at a low VAF in his nail sample (4% in nail vs 23% in blood), suggesting the presence of blood contamination. On subsequent samples, nail clippings were rinsed with water prior to DNA purification, and no MPN driver mutations were detected in washed nails (Patients #2-5). However, since processing of nails for DNA purification is time consuming and laborious, and the DNA yield from nails is quite limited, we are investigating alternative non-invasive sources for germline samples including nasal swabs.Sequencing of matched blood and saliva was performed from patients with all three MPN driver mutations, and in a patient with Clonal Cytopenia of Undetermined Significance (CCUS). All somatic mutations identified in the blood were also seen in the saliva, almost all had identical VAFs from both sample sources, even with VAFs as low as 3%.ConclusionNail clippings are a feasible source for germline DNA in patients with hematologic malignancies and clonal hematopoiesis. Saliva is equivalent to peripheral blood for identifying somatic mutations in hematopoietic cells. These methods of sample collection are non-invasive, amenable to in home collection, and are temperature stable. Collection of saliva and nails could be easily utilized for remotely administered studies by mailing collection kits to participants, thus avoiding the cost and labor of a blood draw. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A rare PALB2 germline variant causing G2/M cell cycle arrest is associated with isolated myelosarcoma in infancy

BackgroundIsolated myelosarcoma of infancy is a rare presentation of acute myelogenous leukaemia (AML). Because of its rarity and early onset in infancy underlying genetic predisposition is potentially relevant in disease initiation.Methods and ResultsWe report an oncologic emergency in an infant with thoracic and intraspinal aleukaemic myeloid sarcoma causing acute myelon compression and lower leg palsy. Whole‐exome sequencing of the patient's germline DNA identified a rare PALB2 (OMIM 610355) variant (p.A1079S), which is located in a domain critical for the gene's proper function within the homology‐directed repair pathway. In line with potential DNA damage repair defects mediated by the PALB2 deregulation, the patient's fibroblasts showed increased sensitivity towards radiation and DNA intercalating agents.ConclusionTherefore, we suggest PALB2 p.A1079S as a pathogenic variant potentially contributing to the here observed patient phenotype.

Polygenic risk score-analysis of thromboembolism in patients with acute lymphoblastic leukemia

IntroductionThromboembolism (TE) is a common and serious toxicity of acute lymphoblastic leukemia (ALL) treatment, but studies of genetic predisposition have been underpowered with conflicting results. We tested whether TE in ALL and TE in the general adult population have a shared genetic etiology. Materials and methodsWe prospectively registered TE events and collected germline DNA in patients 1.0–45.9 years in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL2008 study (7/2008–7/2016). Based on summary statistics from two large genome-wide association studies (GWAS) on venous TE in adults (the International Network of VENous Thromboembolism Clinical Research Networks (INVENT) consortium and the UK Biobank), we performed polygenic risk score (PRS) analysis on TE development in the NOPHO cohort, progressively expanding the PRS by increasing the p-value threshold of single nucleotide polymorphism (SNP) inclusion. Results and conclusionEighty-nine of 1252 patients with ALL developed TE, 2.5 year cumulative incidence 7.2%. PRS of genome-wide significant SNPs from the INVENT and UK Biobank data were not significantly associated with TE, HR 1.16 (p 0.14) and 1.02 (p 0.86), respectively. Expanding PRS by increasing p-value threshold did not reveal polygenic overlap. However, subgroup analysis of adolescents 10.0–17.9 years (n = 231), revealed significant polygenic overlap with the INVENT GWAS. The best fit PRS, including 16,144 SNPs, was associated with TE with HR 1.76 (95% CI 1.23–2.52, empirical p-value 0.02). Our results support an underlying genetic predisposition for TE in adolescents with ALL and should be explored further in future TE risk prediction models.

Case report: recurrent pituitary adenoma has increased load of somatic variants

BackgroundPituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. In this study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in the same patient to explain the causality mechanisms of PA recurrence.Case presentationThe patient was a 69-year-old female with non-functional pituitary macroadenoma with extension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August 2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knosp grade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In both tumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germline DNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling were performed followed by variant filtering and manual review with IGV software. We observed an increased load of missense variants in the recurrent PA tumour when compared to the original tumour. The number of detected variants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally, targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encoded proteins.ConclusionsIn this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression. The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused by clonal expansion of the leftover tumour tissue.

Prevalence of Inherited Cancer Predisposition Mutations in a Cohort of Older AML Patients Enrolled on the Beat AML Master Trial

Prevalence of Inherited Cancer Predisposition Mutations in a Cohort of Older AML Patients Enrolled on the Beat AML Master Trial

Whole Exome Sequencing of Cell-Free DNA for Early Lung Cancer: A Pilot Study to Differentiate Benign From Malignant CT-Detected Pulmonary Lesions.

Introduction: Indeterminate pulmonary lesions (IPL) detected by CT pose a significant clinical challenge, frequently necessitating long-term surveillance or biopsy for diagnosis. In this pilot investigation, we performed whole exome sequencing (WES) of plasma cell free (cfDNA) and matched germline DNA in patients with CT-detected pulmonary lesions to determine the feasibility of somatic cfDNA mutations to differentiate benign from malignant pulmonary nodules.Methods: 33 patients with a CT-detected pulmonary lesions were retrospectively enrolled (n = 16 with a benign nodule, n = 17 with a malignant nodule). Following isolation and amplification of plasma cfDNA and matched peripheral blood mononuclear cells (PBMC) from patient blood samples, WES of cfDNA and PBMC DNA was performed. After genomic alignment and filtering, we looked for lung-cancer associated driver mutations and next identified high-confidence somatic variants in both groups.Results: Somatic cfDNA mutations were observed in both groups, with the cancer group demonstrating more variants than the benign group (1083 ± 476 versus 553 ± 519, p < 0.0046). By selecting variants present in >2 cancer patients and not the benign group, we accurately identified 82% (14/17) of cancer patients.Conclusions: This study suggests a potential role for cfDNA for the early identification of lung cancer in patients with CT-detected pulmonary lesions. Importantly, a substantial number of somatic variants in healthy patients with benign pulmonary nodules were also found. Such “benign” variants, while largely unexplored to date, have widespread relevance to all liquid biopsies if cfDNA is to be used accurately for cancer detection.

Genomic analysis of the origins and evolution of multicentric diffuse lower-grade gliomas

Rare multicentric lower-grade gliomas (LGGs) represent a unique opportunity to study the heterogeneity among distinct tumor foci in a single patient and to infer their origins and parallel patterns of evolution. In this study, we integrate clinical features, histology, and immunohistochemistry for 4 patients with multicentric LGG, arising both synchronously and metachronously. For 3 patients we analyze the phylogeny of the lesions using exome sequencing, including one case with a total of 8 samples from the 2 lesions. One patient was diagnosed with multicentric isocitrate dehydrogenase 1 (IDH1) mutated diffuse astrocytomas harboring distinct IDH1 mutations, R132H and R132C; the latter mutation has been associated with Li-Fraumeni syndrome, which was subsequently confirmed in the patient's germline DNA and shown in additional cases with The Cancer Genome Atlas data. In another patient, phylogenetic analysis of synchronously arising grade II and grade III diffuse astrocytomas demonstrated a single shared mutation, IDH1 R132H, and revealed convergent evolution via non-overlapping mutations in ATRX and TP53. In 2 cases, there was divergent evolution of IDH1-mutated and 1p/19q-codeleted oligodendroglioma and IDH1-mutated and 1p/19q-intact diffuse astrocytoma, occurring synchronously in one case and metachronously in a second. Each tumor in multicentric LGG cases may arise independently or may diverge very early in their development, presenting as genetically and histologically distinct tumors. Comprehensive sampling of these lesions can therefore significantly alter diagnosis and management. Additionally, somatic IDH1 R132C mutation in either multicentric or solitary LGG identifies unsuspected germline TP53 mutation, validating the limited number of published cases.

Tumor testing to identify lynch syndrome in two Australian colorectal cancer cohorts.

Tumor testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was to identify MMR gene mutation carriers in two cohorts of population-based CRC utilizing a combination of tumor and germline testing approaches. Colorectal cancers from 813 patients diagnosed with CRC < 60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry, microsatellite instability (MSI), BRAFV600E somatic mutation, and for MLH1 methylation. MMR gene mutation testing (Sanger sequencing and Multiplex Ligation Dependent Probe Amplification) was performed on germline DNA of patients with MMR-deficient tumors and a subset of MMR-proficient CRCs. Of the 813 ACCFR probands, 90 probands demonstrated tumor MMR deficiency (11.1%), and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR deficiency was identified in the tumors of 103 probands (12.5%) and seven had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 25.2% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively. Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimized by immunohistochemistry screening of CRC diagnosed before 70 years of age. A significant proportion of MMR-deficient CRCs will have unknown etiology (Lynch-like) proving problematic for clinical management.

Genetic variants associated with colorectal brain metastases susceptibility and survival.

Colorectal brain metastases (BM) are rare (1-2%) and a late-stage disease manifestation. Molecular mechanisms for BM development are not well understood. We tested whether variants within genes involved in overcoming the blood-brain barrier (BBB) are associated with BM susceptibility and survival in patients with BM. Germline single-nucleotide polymorphisms (SNPs, n=17) in seven genes (CXCR4, MMP9, ST6GALNAC5, ITGAV, ITGB1, ITGB3, KLF4) were analyzed from germline DNA in patients with resected BM (n=70) or no clinical evidence of BM after at least 24 months from diagnosis (control group, n=45). SNPs were evaluated for association with BM susceptibility and overall survival (OS) from BM diagnosis. ST6GALNAC5 rs17368584 and ITGB3 rs3809865 were significantly associated with BM susceptibility. In multivariable analysis adjusted for patient characteristics, KLF4 rs2236599, ITGAV rs10171481, ST6GALNAC5 rs1883778, CXCR4 rs2680880 and ITGB3 rs5918 were significant for OS. This study shows for the first time that variants within genes involved in breaching the BBB are associated with BM susceptibility and survival. These findings warrant further validation to develop better screening guidelines and to identify novel therapy targets for patients with BM.

MPTH-31CO-EXISTENCE OF TWO TERT PROMOTER REGION SOMATIC POINT MUTATIONS IN A RECURRENT GLIOBLASTOMA

BACKGROUND: Mutations in the promoter region of the telomerase reverse transcriptase gene (TERT) occur at a frequency of greater than 80% in primary GBM. The two most observed TERT promoter mutations in primary GBM, C250T and C228T, each create a de novo transcription factor consensus binding site, upregulating TERT mRNA expression, and are thought to occur in a mutually exclusive fashion. Here we describe the case of a recurrent GBM found to harbor both the C250T and C228T mutations. METHODS: A 57-year-old woman who presented with right hemiparesis and expressive dysphasia was found to have a left frontal contrast-enhancing tumor. She underwent subtotal resection, which revealed primary GBM, and went on to receive standard chemoradiation combined with intra-arterial and intravenous bevacizumab as part of an experimental protocol. She returned with disease progression ten months later and underwent second resection. Fresh tumor was preserved for histopathology, DNA extraction, and was cultured in both serum-free and adherent conditions. RESULTS: Sequencing of tumor DNA revealed that almost all cells harbored the C250T mutation, while a small fraction of cells harbored the C228T mutation. In adherent conditions at early passage, the C250T mutation continued to predominate while there was slight enrichment of C228T. Early and late passage spheres uniformly exhibited the C228T mutation, while C250T was undetectable. Late passage adherent cells exhibited only C228T and could form spheres when transferred into serum-free conditions. All cultures were genotyped and compared to the patient's germline DNA to exclude contamination. CONCLUSIONS: Our results suggest that ongoing convergent evolution gave rise to the C228T mutation in a clone of tumor cells that did not harbor the prevailing C250T mutation of the original tumor. The clone containing C228T exhibited greater proliferation and superior sphere-forming ability in vitro compared to clones containing C250T, and may have been selected through treatment.

A decision support framework for genomically informed investigational cancer therapy.

Rapidly improving understanding of molecular oncology, emerging novel therapeutics, and increasingly available and affordable next-generation sequencing have created an opportunity for delivering genomically informed personalized cancer therapy. However, to implement genomically informed therapy requires that a clinician interpret the patient's molecular profile, including molecular characterization of the tumor and the patient's germline DNA. In this Commentary, we review existing data and tools for precision oncology and present a framework for reviewing the available biomedical literature on therapeutic implications of genomic alterations. Genomic alterations, including mutations, insertions/deletions, fusions, and copy number changes, need to be curated in terms of the likelihood that they alter the function of a "cancer gene" at the level of a specific variant in order to discriminate so-called "drivers" from "passengers." Alterations that are targetable either directly or indirectly with approved or investigational therapies are potentially "actionable." At this time, evidence linking predictive biomarkers to therapies is strong for only a few genomic markers in the context of specific cancer types. For these genomic alterations in other diseases and for other genomic alterations, the clinical data are either absent or insufficient to support routine clinical implementation of biomarker-based therapy. However, there is great interest in optimally matching patients to early-phase clinical trials. Thus, we need accessible, comprehensive, and frequently updated knowledge bases that describe genomic changes and their clinical implications, as well as continued education of clinicians and patients.

Abstract 4280: Integrative analyses of multi-omics sequencing data to guide treatment decisions in a patient with double malignancy

Abstract Multiple primary malignancies are becoming common. The number of second or higher-order cancers is burgeoning and accounted for about 16% of incident cancers. Double malignancy cases pose the problem of finding the best treatment for the patient. Next generation sequencing (NGS) was used in this study to characterize the genetic and genomic composition in a patient with multiple cancer syndromes. A lung adenocarcinoma brain metastasis sample (FFPE) naïve to any tumor therapy, and relapsed primary lung adenocarcinoma and ovarian serous carcinoma tumor biopsies (fresh frozen) were procured from a 51-year old Chinese female never-smoker. The primary lung and ovarian cancer samples were post to the treatments with both first line and EGFR-TKI targeted therapy. Multiple NGS approaches were conducted on the patient biopsies, including DNA whole genome/exome sequencing, RNA sequencing, and microRNA sequencing. Integrative analyses of the comprehensive NGS data indicated EGFR exon 19 deletions in-frame, which encode part of the kinase domain presented in both primary lung cancer and its brain metastases. EGFR T790M mutation which accounts for the resistance of lung tumor to the EGFR-TKI therapy was identified in the relapsed lung sample but not in the brain malignancy. These results were well aligned with the clinical response to EGFR-TKI treatment in the patient. Furthermore, we uncovered frequent occurrences of somatic copy number variations in both lung and ovarian tumors. A somatic non-sense mutation (COSMIC ID: 307331) in TP53 was uniquely identified in the ovarian tumor biopsy, suggesting a different mechanism driving tumorigenesis than that in the lung. Besides, we also identified a heterozygous insertion in DUSP16 and a stop code gained mutation in MAP3K15 in the patient's germline DNA, a potential predisposition of multiple cancer syndromes and early onset of tumor in this patient. Finally, structural variation and gene fusion, gene interaction, pathway analysis of the affected genes were integrated and evaluated. Overall, NGS approaches shed insights into the molecular phenotypes of tumors and thus have the potential to provide the physician key scientific rationale to design targeted therapeutic approaches in the treatment course. Citation Format: Wei Zhu, Jiaqi Huang, Brandon W. Higgs, Philip Brohawn, Shujun Dai, Laura Richman, Bahija Jallal, Liyan Jiang, Yihong Yao. Integrative analyses of multi-omics sequencing data to guide treatment decisions in a patient with double malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4280. doi:10.1158/1538-7445.AM2014-4280

Two single nucleotide polymorphisms in the von Hippel-Lindau tumor suppressor gene in Taiwanese with renal cell carcinoma.

BackgroundRenal cell carcinoma, a common malignant tumor arising from the kidney, occurs in 3.62 and 1.95 cases per one hundred thousand people among men and women, respectively, in Taiwan each year. Approximately 80% of cases are classified as clear-cell renal cell carcinoma. Inactivation of the von Hippel-Lindau tumor suppressor gene has been implicated in the tumorigenic pathway of renal cell carcinoma. Two single nucleotide polymorphisms, rs779805 and rs1642742, located in the promoter and 3′ untranslated regions of the von Hippel-Lindau gene are informative and implicated in the occurrence of renal cell carcinoma worldwide. The aim of this study is to clarify whether these polymorphisms are associated with renal cell carcinoma in Taiwanese. Genomic DNA was isolated from normal and tumor tissues of 19 renal cell carcinoma patients. The samples were screened for allelic polymorphisms by restriction fragment length polymorphism with BsaJ I and Acc I digestion. Reconfirmation was carried out by direct sequencing.ResultsConsistent with Knudson’s two-hit theory, AA to AG somatic mutations were observed in rs779805. In addition, loss of heterozygosity in both rs779805 and rs1642742 was demonstrated in 10 out of 15 RCC patients aged 50 or over. The G allele or AG heterozygote frequencies at these two loci were much higher in patient germline DNA when compared with the control group. After adjusting for age, the frequency of the G allele in both loci was much higher for late onset renal cell carcinoma in the Taiwanese population.ConclusionsOur current results confirmed that the existence of G allele in both rs779805 and rs1642742 in the von Hippel-Lindau tumor suppressor gene is of importance in renal cell carcinoma tumorigenesis. However, more comprehensive and detailed research is needed to address the clinical relevance. Larger sample size is required to determine the exact power of correlation between these two genetic polymorphisms and renal cell carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-638) contains supplementary material, which is available to authorized users.

Tumor Mismatch Repair Immunohistochemistry and DNA MLH1 Methylation Testing of Patients With Endometrial Cancer Diagnosed at Age Younger Than 60 Years Optimizes Triage for Population-Level Germline Mismatch Repair Gene Mutation Testing

Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation.

Epigenetic Deregulation In Relapsed Acute Myeloid Leukemia

Treatment failure in Acute Myeloid Leukemia (AML) is attributed in many cases to relapsed disease. Relapsed AML is a fundamental clinical challenge since most patients have poor clinical outcomes. The exact biological basis of AML relapse remains unclear. Genetic clonal evolution is widely believed to underlie the emergence of chemotherapy resistant clones. However, only limited, predominantly non-overlapping, somatic mutations and copy number aberrations were found to occur upon AML relapse. Furthermore, in a subset of cases, no relapse specific somatic mutations or copy number aberrations were identified. This suggests a role for other mechanisms in relapsed AML. We hypothesize that epigenetic plasticity and deregulation contributes to the pathogenesis of relapse in AML.To explore this notion, we performed a genome scale epigenetic and genetic analysis of thirty-nine paired diagnosis and relapsed AML human patient samples using exome capture, RNA-seq and ERRBS for DNA methylation sequencing. Exome capture was performed on each patient's germline DNA as well. Exome capture revealed only a limited number of known recurrent somatic mutations acquired upon disease relapse, in agreement with previous reports. In contrast, upon disease relapse we identified thousands of statistically significant changes in cytosine methylation patterns. Globally, the majority of patients (85%) displayed striking predominance of DNA hypermethylation (p= 1.00433e-05, binomial test for equality of proportions) upon disease relapse. Notably a smaller set of patients displayed the opposite epigenetic phenotype with prominent loss of cytosine methylation. While differential methylation in the hypermethylated group of patients localized predominantly to CpG islands, the majority of differential methylation in the hypomethylated group localized to regions lacking both CpG islands and shores. In spite of these two distinct overall cytosine methylation patterns, the majority of differentially methylated cytosines are located in intergenic regions in all cases, and a subset of promoters were hypermethylated in almost all patients at relapse. A pathway analysis indicated that the commonly hypermethylated gene promoters at relapse are involved in the Hedghog, Wnt and calcium signaling pathways (p<0.05, modified Fisher Exact test). Integration of these findings with mutational and transcriptional profiles is underway.In order to determine whether epigenetic events linked to AML relapse could be modeled experimentally we performed a pilot study of a human AML xenograft in immunocompromised mice. Engrafted mice were treated with Ara-C at a clinically relevant dose (60mg/Kg; n=2) or vehicle alone (n=3) for five consecutive days. Human AML cells were collected at various timepoints including 28 days after Ara-C treatment where the AML had frankly relapsed in mice. Cytosine methylation profiles obtained through ERRBS revealed predominantly hypermethylated cytosines when compared to the xenotransplanted diagnostic sample (72% hypermethylated versus 28% hypomethylated). Remarkably, there was a strong overlap with gene promoters that are also aberrantly methylated in relapsed AML patients (p<0.01, hypergeometric test), including members of the Wnt signaling pathway.We conclude that there are epigenetically distinct forms of relapsed AML. Nonetheless, there is convergent epigenetic regulation of specific gene pathways that may contribute to relapsed AML pathogenesis and xenotransplanted AML mice can serve as experimental models for further study. Finally, the genomic distribution of reprogrammed methylation suggests a role for epigenetic plasticity at distal regulatory elements. Whereas it remains unclear whether these changes represent clonal selection, their extensive and dynamic range suggest that exposure to chemotherapy may alter the fidelity of mechanisms that control cytosine methylation distribution thus permitting widespread and distant epigenetic reprogramming and contributing to disease relapse. Disclosures:No relevant conflicts of interest to declare.

AXIN genetic analysis in adrenocortical carcinomas updated.

Wnt/β-catenin signaling pathway activation plays an important role in adrenocortical tumorigenesis, but is only in part related to β-catenin activating somatic mutations. Recently, genetic alteration in AXIN2, a key component of the Wnt/β-catenin signaling pathway, has been described in adrenocortical tumors and specifically in adrenocortical carcinoma (ACC). To assess frequency and consequences of AXIN genes alteration on a large cohort of ACC. Forty-nine adult sporadic ACC, with expression data available, in addition to both ACC cell lines H295 and H295R were studied. AXIN2 exon 8 hot-spot sequencing was performed on the entire cohort. AXIN1 entire coding region was studied on the 8 ACC with nuclear β-catenin staining. The previously described AXIN2 in-frame heterozygous 12bp deletion c2013_2024del12 was found in 1 of the 49 ACC studied (2%), in a tumor with pSer45del activating CTNNB1 mutation and nuclear β-catenin staining. This heterozygous deletion was also found in the patient's germline DNA, extracted from peripheral blood leukocytes. This genetic alteration was also present in H295 and H295R cell lines. The single-nucleotide polymorphism rs35415678 was found with an allele frequency similar to those found in reference populations. No correlation between AXIN2 expression, AXIN2 genetic variant or nuclear β- catenin staining was observed. No AXIN1 alterations were found in the 8 ACC studied. AXIN genes do not play a major role in ACC tumorigenesis and Wnt/β-catenin signaling pathway activation. AXIN2 germline variant c2013_2024del12 is likely to be a non-pathogenic polymorphism.

Identification and functional characterization of a novel MUTYH gene mutation.

e12026 Background: Biallelic germline mutations in MUTYH result in the autosomal recessive syndrome of MUTYH associated polyposis (MAP).Three well-known, common mutations account for the vast majority of identifiable germline mutations, and serve as the basis for current genetic testing strategies. Comprehensive sequencing of MUTYH often identifies variants of uncertain pathologic significance, and studies to determine the pathogenicity of newly identified variants may offer valuable clinical information and mechanistic insights. In the present study we seek to describe the base-excision repair function of a novel MUTYH (p.C306W) mutation identified in a patient with multiple colon polyps and a family history of colon cancers. Methods: A 50 year old patient with &gt;50 adenomas underwent clinical and laboratory evaluation to assess for germline genetic mutations. We performed Sanger sequencing of tumor and germline DNA together with targeted restriction enzyme digest of germline DNA and fragment DNA sequencing of the alleles. We prepared MUTYH proteins with protein liquid chromatography and assessed their mismatched adenine excision repair capacity employing a glycosylase assay. Results: Analysis of the patient's germline DNA revealed an absence of APC mutations, and the presence of the previously well characterized p.G396D MUTYH mutation as well as a novel p.C306W mutation. Targeted restriction enzyme digest demonstrated trans configuration of the p.G396D and p.C306W MUTYH mutations. Mismatched adenine excision functionality of wildtype MUTYH, known mutant controls p.G396D and p.Y179C, and putative mutant p.C306W were assessed in the glycosylase assay. Consistent with previous experimental observations, relative to wildtype MUTYH, the p.G396D and p.Y179C MUTYH mutants demonstrated attenuated adenine excision activities of 43% and 0%, respectively. Comparable to the activity of the pY179C mutant, the novel p.C306D mutant demonstrated 0% adenine excision activity. Subsequent tumor analysis demonstrated G:C to T:A transversion in the APC gene in somatic DNA derived from an adenoma. Conclusions: Experimental and clinical data demonstrate that p.C306D MUTYH is a pathogenic mutation contributing to the phenotype of MAP.

Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in germline DNA of patients with polycythemia

Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma. Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively. This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category. As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.

Sporadic breast cancer patients' germline DNA exhibit an AT‐rich microsatellite signature

Using a custom CGH-like oligonucleotide array to measure the global microsatellite content in the genomes of 72 cancer, cancer-free, and high risk patient and cell line samples (56 germline DNA and 16 in tumor or tumor cell line DNA) we found a unique, reproducible, and statistically significant pattern of 18 motif-specific microsatellite families (out of 962 possible 1-6 mer repeats) in breast cancer patient germline and tumor DNA, but not in germline DNA of cancer-free volunteer controls or in breast cancer patients with BRCA1/2 mutations. These high-similarity A/T rich repetitive motifs were also more pronounced in the germlines and tumors of colon cancer tumor patients (3/6 samples) and microsatellite unstable colon cancer cell lines; however, germline DNA of sporadic breast cancer patients exhibited the largest global content shift for those motifs with extreme AT/GC ratios. These results indicate that global microsatellite variability is complex, suggest the existence of a previously unknown genomic destabilization mechanism in breast cancer patients' germline DNA, and warrant further testing of such microsatellite variability as a predictor of future breast cancer development.