Glioblastoma multiforme and medulloblastoma are the most frequent high-grade brain tumors in adults and children, respectively. Standard therapies for these cancers are mainly based on surgical resection, radiotherapy, and chemotherapy. However, intrinsic or acquired resistance to treatment occurs almost invariably in the first case, and side effects are unacceptable in the second. Therefore, the development of new, effective drugs is a very important unmet medical need. A critical requirement for developing such agents is to identify druggable targets required for the proliferation or survival of tumor cells, but not of other cell types. Under this perspective, genes mutated in congenital microcephaly represent interesting candidates. Congenital microcephaly comprises a heterogeneous group of disorders in which brain volume is reduced, in the absence or presence of variable syndromic features. Genetic studies have clarified that most microcephaly genes encode ubiquitous proteins involved in mitosis and in maintenance of genomic stability, but the effects of their inactivation are particularly strong in neural progenitors. It is therefore conceivable that the inhibition of the function of these genes may specifically affect the proliferation and survival of brain tumor cells. Microcephaly genes encode for a few kinases, including CITK, PLK4, AKT3, DYRK1A, and TRIO. In this review, we summarize the evidence indicating that the inhibition of these molecules could exert beneficial effects on different aspects of brain cancer treatment.

RATIONALE: Holoprosencephaly (HPE) is a severe congenital brain malformation resulting from failed or incomplete forebrain division in early pregnancy.
PATIENT CONCERNS: In this study, we reported a 9-month old infant girl with mild microcephaly, semilobor HPE, and arachnoid cyst.
DIAGNOSES: Potential genetic defects were screened directly using trio-case whole exome sequencing (WES) rather than traditional karyotype, microarray, and Sanger sequencing of select genes.
OUTCOMES: A previous unpublished de novo missense mutation (c.1069C >G, p.H357D) in the 3rd zinc finger domain (ZFD3) of the ZIC2 gene was identified in the affected individual, but not in the parents. Sanger sequencing using specific primers verified the mutation. Extensive bioinformatics analysis confirmed the pathogenicity of this extremely rare mutation. Phenotype-genotype analysis revealed significant correlation between the 3rd zinc-finger domain with semilobor HPE.
LESSONS: These findings expanded the spectrum of the ZIC2 gene mutations and associated clinical manifestations, which is the first identification of a mutated ZIC2 gene in a Han infant girl with mild microcephaly, semilobor HPE, and arachnoid cyst.

BACKGROUND: We describe a patient presenting with pachygyria, epilepsy, developmental delay, short stature, failure to thrive, facial dysmorphisms, and multiple osteochondromas.
METHODS: The patient underwent extensive genetic testing and analysis in an attempt to diagnose the cause of his condition. Clinical testing included metaphase karyotyping, array comparative genomic hybridization, direct sequencing and multiplex ligation-dependent probe amplification and trio-based exome sequencing. Subsequently, research-based whole transcriptome sequencing was conducted to determine whether it might shed light on the undiagnosed phenotype.
RESULTS: Clinical exome sequencing of patient and parent samples revealed a maternally inherited splice-site variant in the doublecortin (DCX) gene that was classified as likely pathogenic and diagnostic of the patient's neurological phenotype. Clinical array comparative genome hybridization analysis revealed a 16p13.3 deletion that could not be linked to the patient phenotype based on affected genes. Further clinical testing to determine the cause of the patient's multiple osteochondromas was unrevealing despite extensive profiling of the most likely causative genes, EXT1 and EXT2, including mutation screening by direct sequence analysis and multiplex ligation-dependent probe amplification. Whole transcriptome sequencing identified a SAMD12-EXT1 fusion transcript that could have resulted from a chromosomal deletion, leading to the loss of EXT1 function. Re-review of the clinical array comparative genomic hybridization results indicated a possible unreported mosaic deletion affecting the SAMD12 and EXT1 genes that corresponded precisely to the introns predicted to be affected by a fusion-causing deletion. The existence of the mosaic deletion was subsequently confirmed clinically by an increased density copy number array and orthogonal methodologies CONCLUSIONS: While mosaic mutations and deletions of EXT1 and EXT2 have been reported in the context of multiple osteochondromas, to our knowledge, this is the first time that transcriptomics technologies have been used to diagnose a patient via fusion transcript analysis in the congenital disease setting.

BACKGROUND/AIM: The aim of this study was to evaluate the usefulness of biomarkers related to prostate cancer metastasis and survival of patients.
MATERIALS AND METHODS: Proteomics were used for detecting significant differences in protein expression among normal prostate, localized prostate cancer and metastatic cancer using 2-dimensional gel electrophoresis and mass spectrometry. mRNA expression was then examined in order to further confirm significant differences in protein expression. A total of 7 proteins were found to be differentially expressed. Immunochemistry (IHC), was also used to confirm the levels of expression of the 7 proteins in prostate cancer. Survival analysis using the candidate markers was finally performed in 98 metastatic prostate cancer patients according to IHC results.
RESULTS: In metastatic lesions, proteomic analysis indicated that heat shock protein (HSP) 27, prohibitin, glutathione S-transferase 1, fibrinogen β chain, and aldehyde dehydrogenase 6A1 were up-regulated, while α1 antitrypsin, and HSP 60 were down-regulated. IHC revealed that HSP 27, ALDH6A1 and prohibitin were highly specific to metastatic tumor cells. HSP27 and prohibitin appeared more strongly in the incipient stage of cancer than metastatic cancer, and ALDH6A1 was significantly reduced in metastatic cancer (p<0.01). Of all proteins, phohibitin had the highest value in predicting survival. However, all three proteins were a stronger marker than each one separately.
CONCLUSION: Trio-biomarker composed of HSP27, ALDH6A1 and prohibitin may predict survival of metastatic prostate cancer patients.

The discovery of cancer-predisposing syndromes (CPSs) using next-generation sequencing (NGS) technologies is of increasing importance in pediatric oncology with regard to diagnosis, treatment, surveillance, family counselling and research. Recent studies indicate that a considerable percentage of childhood cancers are associated with CPSs. However, the ratio of CPSs that are caused by inherited vs. de novo mutations (DNMs), the risk of recurrence, and even the total number of genes, which should be considered as a true cancer-predisposing gene, are still unknown. In contrast to sequencing only single index patients, family-based NGS of the germline is a very powerful tool for providing unique insights into inheritance patterns (e.g., DNMs, parental mosaicism) and types of aberrations (e.g., SNV, CNV, indels, SV). Furthermore, functional perturbations of key cancer pathways (e.g., TP53, FA/BRCA) by at least two co-inherited heterozygous digenic mutations from each parent and currently unrecognized rare variants and unmeasured genetic interactions between common and rare variants may be a widespread genetic phenomenon in the germline of affected children. Therefore, family-based trio sequencing has the potential to reveal a striking new landscape of inheritance in childhood cancer and to facilitate the integration and efforts of individualized treatment strategies, including personalized and preventive medicine and cancer surveillance programs. Consequently, cancer genetics is becoming an increasingly common approach in modern oncology, so trio-sequencing should also be routinely integrated into pediatric oncology.

BACKGROUND: Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations.
METHODS: In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published "nontumor"-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined.
RESULTS: Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in "nontumor" tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in "paratumor" tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from "nontumor" to "paratumor" and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations.
CONCLUSIONS: These findings suggest that developing cancer cells undergo sequential changes that enable the "nontumor" cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the "paratumor" cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

Metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria (MC-HGA) (OMIM 614875) is a severe chondrodysplasia combined with a urinary excretion of D-2-hydroxyglutaric acid. Here, we reported the tenth case of this disease. A 15-year-old boy had symmetric radiolulencies in the metaphyses of the long bones suggesting enchondromatosis and platyspondyly. Remarkably, he manifested widespread cavernous hemangiomas including scalp, lips, tongue, larynx, and prepuce, with the onset of 3 years of age. Hemangiomas at the larynx had caused dyspnea and those in the oral cavity led to recurrent bleeding, requiring several surgical removals. These multiple and debilitating hemangiomas have never been previously reported in patients with MC-HGA. Mutation analyses including Sanger sequencing of genes involving in enchondromatosis and the metabolic pathway of D-2-hydroxyglutarate including PTHR1, D2HGDH, HOT, and IDH1, as well as whole-exome sequencing for proband-parent trio analysis and paired blood versus hemangioma studies showed no pathogenic variants. In summary, we reported the tenth patient with MC-HGA who manifested widespread and debilitating hemangiomas in several organs, expanding the clinical spectrum of MC-HGA.

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors.
METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed.
RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma.
CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

This is a review of the morphological spectrum of fatty tumours containing a component of spindle cells, highlighting the immunohistochemical and cytogenetic workup that is now mandatory for accurate diagnosis, with the goal of providing a practical approach for practising surgical pathologists. There have been significant advances in recent years in classifying and understanding the pathogenesis of fatty tumours with spindle cells, based on the correlation of histological, immunohistochemical and cytogenetic/molecular findings. In spite of this, morphological diagnosis and accurate classification of fatty tumours with spindle cells can be challenging to diagnostic pathologists. A group of three lesions: spindle cell lipoma, mammary-type myofibroblastoma and cellular angiofibroma share morphological features and are united by retinoblastoma protein (pRb) loss. Closely allied to these lesions, especially spindle cell lipoma is the newly designated atypical spindle cell lipomatous tumour, which shares morphological, immunohistochemical and cytogenetic features with the trio of tumours lacking nuclear pRb. All of these lesions lack

Triple functional domain protein (Trio) is an evolutionarily conserved protein with guanine nucleotide exchange factors that regulate different physiological processes in some types of cancer. However, the expression and function of Trio in cervical cancer are still unknown. The purpose of this study was to detect the expression of Trio in cervical cancer tissues and to evaluate its clinical value. Furthermore, the effects of the Trio on the migration and invasion of cervical cancer cells and its mechanism were investigated in vitro. The results of the present study revealed that Trio expression levels were significantly higher in most of the clinical cervical cancer samples than in adjacent tissues. The clinicopathological significance of Trio expression was also analyzed, and the results revealed that high expression levels in cervical cancer were correlated with lymph node metastasis (P=0.005). The CRISPR/Cas9 system was used to knockdown the endogenous Trio. The inhibition of Trio significantly decreased the migration and invasion abilities of cervical cancer cells. Meanwhile, levels of RhoA/ROCK signaling factors (RhoA, Rock, and p-LIMK), which contributed to cell migration and invasion, were decreased along with the inhibition of Trio. Therefore, Trio may regulate the migration and invasion of cervical cancer through the RhoA/ROCK signaling pathway.

An accurate tool enabling early diagnosis of hepatocellular carcinoma (HCC) is clinically important, given that early detection of HCC markedly improves survival. We aimed to investigate the molecular markers underlying early progression of HCC that can be detected in precancerous lesions. We designed a gene selection strategy to identify potential driver genes by integrative analysis of transcriptome and clinicopathological data of human multistage HCC tissues, including precancerous lesions, low- and high-grade dysplastic nodules. The gene selection process was guided by detecting the selected molecules in both HCC and precancerous lesion. Using various computational approaches, we selected 10 gene elements as a candidate and, through immunohistochemical staining, showed that barrier to autointegration factor 1 (BANF1), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), and splicing factor 3b subunit 4 (SF3B4) are HCC decision markers with superior capability to diagnose early-stage HCC in a large cohort of HCC patients, as compared to the currently popular trio of HCC diagnostic markers: glypican 3, glutamine synthetase, and heat-shock protein 70. Targeted inactivation of BANF1, PLOD3, and SF3B4 inhibits in vitro and in vivo liver tumorigenesis by selectively modulating epithelial-mesenchymal transition and cell-cycle proteins. Treatment of nanoparticles containing small-interfering RNAs of the three genes suppressed liver tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. We also demonstrated that SF3B4 overexpression triggers SF3b complex to splice tumor suppressor KLF4 transcript to nonfunctional skipped exon transcripts. This contributes to malignant transformation and growth of hepatocyte through transcriptional inactivation of p27
CONCLUSION: The findings suggest molecular markers of BANF1, PLOD3, and SF3B4 indicating early-stage HCC in precancerous lesion, and also suggest drivers for understanding the development of hepatocarcinogenesis. (Hepatology 2018;67:1360-1377).

Li-Fraumeni syndrome is a clinically heterogeneous familial cancer predisposition syndrome with autosomal-dominant inheritance caused by heterozygous germline mutations in the TP53 gene. We here analyze the genetic background of a family with a 4-year-proband presented with a Li-Fraumeni tumor. The mother developed breast cancer at age 37 and the proband died at age 8. We performed Sanger sequencing and whole-exome sequencing on peripheral blood DNA from proband and relatives. Data analysis selected only high-quality score and depth reads, rare variants and protein impact involving missense, non-sense, frameshift and splice disrupt mutations. Disease implicated variants and predicted deleterious alterations were also chosen. TP53 genetic testing revealed a never reported TP53 deletion arose as de novo mutation in the mother and inherited by the proband. We then performed whole-exome analysis of the trio to uncover inherited variants from the father that potentially worsen the already altered genetic background in the proband. No pathogenic variants were inherited in autosomal recessive, de novo dominant or X-linked recessive manner. Comparing proband and father exome we detected 25 predicted deleterious variants including a nonsense mutation in ERCC3. Those inherited mutations are possible candidate modifiers linked to TP53, explaining the proband accelerated tumor onset compared to the mother and providing a possible explanation of the genetic anticipation event in this Li-Fraumeni family.

We conducted an exome-wide association study of childhood acute lymphoblastic leukemia (ALL) among Hispanics to confirm and identify novel variants associated with disease risk in this population. We used a case-parent trio study design; unlike more commonly used case-control studies, this study design is ideal for avoiding issues with population stratification bias among this at-risk ethnic group. Using 710 individuals from 323 Guatemalan and US Hispanic families, two inherited SNPs in ARID5B reached genome-wide level significance: rs10821936, RR = 2.31, 95% CI = 1.70-3.14, p = 1.7×10-8 and rs7089424, RR = 2.22, 95% CI = 1.64-3.01, p = 5.2×10-8. Similar results were observed when restricting our analyses to those with the B-ALL subtype: ARID5B rs10821936 RR = 2.22, 95% CI = 1.63-3.02, p = 9.63×10-8 and ARID5B rs7089424 RR = 2.13, 95% CI = 1.57-2.88, p = 2.81×10-7. Notably, effect sizes observed for rs7089424 and rs10821936 in our study were >20% higher than those reported among non-Hispanic white populations in previous genetic association studies. Our results confirmed the role of ARID5B in childhood ALL susceptibility among Hispanics; however, our assessment did not reveal any strong novel inherited genetic risks for acute lymphoblastic leukemia among this ethnic group.

Myxofibrosarcomas are morphologically heterogeneous soft tissue sarcomas lacking a specific immunohistochemical expression profile and recurrent genetic changes. The study was designed to gain further insights into the molecular landscape of myxofibrosarcomas by targeted re-sequencing of known cancer driver hotspot mutations and the analysis of genomewide somatic copy number alterations. A well-defined group of myxofibrosarcomas, including myxofibrosarcomas G1 (n=6), myxofibrosarcomas G3 (n=7), myxofibrosarcomas with morphologically heterogeneous and independently selectable G1 and G3 areas within a tumor (n=8), and myxofibrosarcomas G3 with subsequent tumor recurrence (n=1) or metastatic disease (n=3) were evaluated. Mutational analysis demonstrated mutations in TP53, PTEN, FGFR3, CDKN2A, and RB1. TP53 mutations were seen in 11 (44%) of patients and detected in myxofibrosarcomas G1, G3, with heterogeneous morphology and G3 with subsequent metastases in 1 patient (16%), 3 patients (42%), 2 patients (62.5%), and 3 patients (75%), respectively. Additional mutations were detected in 2 patients, intratumoral mutational heterogeneity in 1 patient. We observed a variety of copy number alterations typical for myxofibrosarcomas, with higher numbers in G3 compared with G1 myxofibrosarcomas. Cluster analysis revealed distinctive features especially in metastatic and recurrent disease. Focal alterations affected CDKN2A, CCND1, CCNE1, EGFR, EPHA3, EPHB1, FGFR1, JUN, NF1, RB1, RET, TP53, and additional novel amplifications in CCNE1, KIT, EGFR, RET, BRAF, NTRK2 were seen in G3 compared with the G1 tumor areas. The total number of focal events in G1 versus G3 tumors differed significantly (P=0.0014). TRIO and RICTOR co-amplification was seen in 8 (44%) G3 and 1 (10%) G1 myxofibrosarcomas and RICTOR amplification alone in 4 (40%) G1 myxofibrosarcomas. TRIO amplification was significantly (P=0.0218) higher in G3 myxofibrosarcomas indicating a late genetic event. These findings support the use of expanded molecular profiling in myxofibrosarcomas to detect drug-able targets to allow patients to participate in basket trials.

BACKGROUND: Lipopolysaccharide-responsive, beige-like anchor protein (LRBA) deficiency is characterized by primary immunodeficiency and autoimmunity. Cancer may present another feature of LRBA deficiency. We describe a case history of a young adult with LRBA deficiency and two independent malignancies.
METHODS: Family-trio whole exome sequencing with unbiased phenotype ontology approach was used for identification of causative mutations of a primary immune deficiency disorder. Additionally, we sought to identify germline mutations in genes known to be associated with two independent malignancies using a targeted approach. A cytotoxic T-lymphocyte associated protein 4 (CTLA4) expression in T lymphocytes was determined by flow cytometry.
RESULTS: In the patient with clinical signs of LRBA deficiency multifocal gastric carcinoma and malignant melanoma were diagnosed and surgically treated at 19 and 27 years of age, respectively. Despite refusal of any adjuvant chemotherapy or radiotherapy, the patient demonstrated disease free survival for at least 13 years after the first cancer diagnosis. A homozygous frameshift deletion in LRBA gene (p.Glu946Ter) and two common variants in TYR gene were identified. Reduced CTLA4 expression in a subset of regulatory T lymphocytes was identified in the patient and his unaffected mother carrying a heterozygous LRBA mutation as compared to control in a dose-dependent manner.
CONCLUSION: This is the first description of gastric cancer and malignant melanoma in a young adult with LRBA deficiency. The role of LRBA gene knockout in cancer development and its prognosis remains to be elucidated.

We report a family in which two brothers had an undiagnosed genetic disorder comprised of dysmorphic features, microcephaly, severe intellectual disability (non-verbal), mild anemia, and cryptorchidism. Both developed osteosarcoma. Trio exome sequencing (using blood samples from the younger brother and both parents) was performed and a nonsense NM_000489.4:c.7156C>T (p.Arg2386*) mutation in the ATRX gene was identified in the proband (hemizygous) and in the mother's peripheral blood DNA (heterozygous). The mother is healthy, does not exhibit any clinical manifestations of ATR-X syndrome and there was no family history of cancer. The same hemizygous pathogenic variant was confirmed in the affected older brother's skin tissue by subsequent Sanger sequencing. Chromosomal microarray studies of both brothers' osteosarcomas revealed complex copy number alterations consistent with the clinical diagnosis of osteosarcoma. Recently, somatic mutations in the ATRX gene have been observed as recurrent alterations in both osteosarcoma and brain tumors. However, it is unclear if there is any association between osteosarcoma and germline ATRX mutations, specifically in patients with constitutional ATR-X syndrome. This is the first report of osteosarcoma diagnosed in two males with ATR-X syndrome, suggesting a potential increased risk for cancer in patients with this disorder.

Genomic mosaicism arising from postzygotic mutations has long been associated with cancer and more recently with non-cancer diseases. It has also been detected in healthy individuals including healthy parents of children affected with genetic disorders, highlighting its critical role in the origin of genetic mutations. However, most existing software for the genome-wide identification of single-nucleotide mosaicisms (SNMs) requires a paired control tissue obtained from the same individual which is often unavailable for non-cancer individuals and sometimes missing in cancer studies. Here, we present MosaicHunter (http://mosaichunter.cbi.pku.edu.cn), a bioinformatics tool that can identify SNMs in whole-genome and whole-exome sequencing data of unpaired samples without matched controls using Bayesian genotypers. We evaluate the accuracy of MosaicHunter on both simulated and real data and demonstrate that it has improved performance compared with other somatic mutation callers. We further demonstrate that incorporating sequencing data of the parents can be an effective approach to significantly improve the accuracy of detecting SNMs in an individual when a matched control sample is unavailable. Finally, MosaicHunter also has a paired mode that can take advantage of matched control samples when available, making it a useful tool for detecting SNMs in both non-cancer and cancer studies.

The fundamental metabolic decision of a cell, the balance between respiration and fermentation, rests in part on expression of the mitochondrial genome (mtDNA) and coordination with expression of the nuclear genome (nuDNA). Previously we described mtDNA copy number depletion across many solid tumor types (Reznik et al., 2016). Here, we use orthogonal RNA-sequencing data to quantify mtDNA expression (mtRNA), and report analogously lower expression of mtRNA in tumors (relative to normal tissue) across a majority of cancer types. Several cancers exhibit a trio of mutually consistent evidence suggesting a drop in respiratory activity: depletion of mtDNA copy number, decreases in mtRNA levels, and decreases in expression of nuDNA-encoded respiratory proteins. Intriguingly, a minority of cancer types exhibit a drop in mtDNA expression but an increase in nuDNA expression of respiratory proteins, with unknown implications for respiratory activity. Our results indicate suppression of respiratory gene expression across many cancer types.

BACKGROUND: Congenital dilatation of the bile-duct (CDD) is a rare, mostly sporadic, disorder that results in bile retention with severe associated complications. CDD affects mainly Asians. To our knowledge, no genetic study has ever been conducted.
METHODS: We aim to identify genetic risk factors by a "trio-based" exome-sequencing approach, whereby 31 CDD probands and their unaffected parents were exome-sequenced. Seven-hundred controls from the local population were used to detect gene-sets significantly enriched with rare variants in CDD patients.
RESULTS: Twenty-one predicted damaging de novo variants (DNVs; 4 protein truncating and 17 missense) were identified in several evolutionarily constrained genes (p < 0.01). Six genes carrying DNVs were associated with human developmental disorders involving epithelial, connective or bone morphologies (PXDN, RTEL1, ANKRD11, MAP2K1, CYLD, ACAN) and four linked with cholangio- and hepatocellular carcinomas (PIK3CA, TLN1 CYLD, MAP2K1). Importantly, CDD patients have an excess of DNVs in cancer-related genes (p < 0.025). Thirteen genes were recurrently mutated at different sites, forming compound heterozygotes or functionally related complexes within patients.
CONCLUSIONS: Our data supports a strong genetic basis for CDD and show that CDD is not only genetically heterogeneous but also non-monogenic, requiring mutations in more than one genes for the disease to develop. The data is consistent with the rarity and sporadic presentation of CDD.

Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390-400. ©2016 AACR.

Metastatic dissemination is the cause of death in the vast majority of cancers, including lung cancers. In order to metastasize, tumor cells must undergo a well-known series of changes, however the molecular details of how they manage to overcome the barriers at each stage remain incomplete. One critical step is acquiring the ability to migrate through the extracellular matrix. Loss of expression of the RAS-related small GTPase RHOB is a common feature of lung cancer progression, and we recently reported that this induces an epithelial-to-mesenchymal transition (EMT) that is dependent on SLUG overexpression and E-Cadherin inhibition and is characterized by 3-dimensional cell shape reorganization and the increased invasiveness of bronchial cells. RHOB loss was found to induce AKT1 activation, which in turn activates RAC1 through its GEF TRIO. Further investigation of this pathway revealed that RHOB interacts with and positively regulates PP2A, one of the major cellular serine-threonine phosphatases, by recruiting its regulatory subunit B55. Here we discuss the role of this newly discovered RHOB/PP2A/AKT1/RAC1 pathway in relation to mesenchymal migration and invasion in lung cancer.

Myxofibrosarcoma is a common mesenchymal malignancy with complex genomics and heterogeneous clinical outcomes. Through gene-expression profiling of 64 primary high-grade myxofibrosarcomas, we defined an expression signature associated with clinical outcome. The gene most significantly associated with disease-specific death and distant metastasis was ITGA10 (integrin-α10). Functional studies revealed that myxofibrosarcoma cells strongly depended on integrin-α10, whereas normal mesenchymal cells did not. Integrin-α10 transmitted its tumor-specific signal via TRIO and RICTOR, two oncoproteins that are frequently co-overexpressed through gene amplification on chromosome 5p. TRIO and RICTOR activated RAC/PAK and AKT/mTOR to promote sarcoma cell survival. Inhibition of these proteins with EHop-016 (RAC inhibitor) and INK128 (mTOR inhibitor) had antitumor effects in tumor-derived cell lines and mouse xenografts, and combining the drugs enhanced the effects. Our results demonstrate the importance of integrin-α10/TRIO/RICTOR signaling for driving myxofibrosarcoma progression and provide the basis for promising targeted treatment strategies for patients with high-risk disease.
SIGNIFICANCE: Identifying the molecular pathogenesis for myxofibrosarcoma progression has proven challenging given the highly complex genomic alterations in this tumor type. We found that integrin-α10 promotes tumor cell survival through activation of TRIO-RAC-RICTOR-mTOR signaling, and that inhibitors of RAC and mTOR have antitumor effects in vivo, thus identifying a potential treatment strategy for patients with high-risk myxofibrosarcoma. Cancer Discov; 6(10); 1148-65. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.

PURPOSE: Despite various differences, nontranslocation-related sarcomas (e.g., comprising undifferentiated pleomorphic sarcoma, leiomyosarcoma, myxofibrosarcoma) are unified by their complex genetics. Extensive analysis of the tumor genome using molecular cytogenetic approaches showed many chromosomal gains, losses, and translocations per cell. Genomic quantitative alterations and expression variations have been extensively studied by adapted high-throughput approaches, yet translocations still remained unscreened. We therefore analyzed 117 nontranslocation-related sarcomas by RNA sequencing to identify fusion genes.
EXPERIMENTAL DESIGN: We performed RNA sequencing and applied a bioinformatics pipeline dedicated to the detection of fusion transcripts. RT-PCR and Sanger sequencing were then applied to validate predictions and to search for recurrence and specificity.
RESULTS: Among the 6,772 predicted fusion genes, 420 were in-frame. One recurrent rearrangement, consistently involving TRIO with various partners, was identified in 5.1% of cases. TRIO translocations are either intrachromosomal with TERT or interchromosomal with LINC01504 or ZNF558 Our results suggest that all translocations led to a truncated TRIO protein either directly or indirectly by alternative splicing. TRIO rearrangement is associated with a modified transcriptomic program to immunity/inflammation, proliferation and migration, and an increase in proliferation.
CONCLUSIONS: TRIO fusions have been identified in four different sarcoma histotypes, likely meaning that they are not related to a primary oncogenic event but rather to a secondary one implicated in tumor progression. Moreover, they appear to be specific to nontranslocation-related sarcomas, as no such rearrangement was identified in sarcomas with simple genetics. More cases could lead to a significant association of these fusions to a specific clinical behavior. Clin Cancer Res; 23(3); 857-67. ©2016 AACR.

The tumor-suppressor RUNX3 has a critical role in a lineage determination, cell cycle arrest and apoptosis. Lozenge (Lz), a Drosophila homolog of mammalian RUNX family members, has integral roles in these processes and specifically in eye cell fate determination. To elucidate the genetic modifiers of Lz/RUNX3, we performed a large-scale functional screen in a fly mutant library. The screen revealed genetic interactions between the Lz, Rac and Hippo pathways. Analysis of interactions among these genes revealed that the defective phenotype resulting from activation of Yki, an end point effector of the Hippo pathway, was suppressed by Lz and enhanced by Rac-Trio. Molecular biological analysis using mammalian homologs reveled that LATS1/2-mediated YAP phosphorylation-facilitated dissociation of the YAP-TEAD4 complex and association of the YAP-RUNX3 complex. When cells were stimulated to proliferate, activated RAC-TRIO signaling inhibited LATS1/2-mediated YAP phosphorylation; consequently, YAP dissociated from RUNX3 and associated with TEAD, thereby replacing the YAP-RUNX3 complex with YAP-TEAD. RUNX3 contributed to both association and dissociation of YAP-TEAD complex, most likely through the formation of the YAP-TEAD-RUNX3 ternary complex. Ectopic expression of RUNX3 in MKN28 gastric cancer cells reduced tumorigenicity, and the tumor-suppressive activity of RUNX3 was associated with its ability to interact with YAP. These results identify a novel regulatory mechanism, mediated by the Hippo and RAC-TRIO pathways, that changes the binding partner of YAP.

BACKGROUND AND AIMS: Elevated circulating concentrations of the hormone gastrin contribute to the development of gastric adenocarcinoma and types-1 and 2 gastric neuroendocrine tumors (NETs). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate proteins which in turn influence various biological processes. We hypothesised that gastrin induces the expression of specific gastric miRNAs within CCK2 receptor (CCK2R) expressing cells and that these mediate functionally important actions of gastrin.
RESULTS: Gastrin increased miR-222 expression in AGSGR cells, with maximum changes observed at 10 nM G17 for 24 h. Signalling occurred via CCK2R and the PKC and PI3K pathways. miR-222 expression was increased in the serum and gastric corpus mucosa of hypergastrinemic INS-GAS mice and hypergastrinemic patients with autoimmune atrophic gastritis and type 1 gastric NETs; it decreased in patients following treatment with the CCK2R antagonist netazepide (YF476). Gastrin-induced miR-222 overexpression resulted in reduced expression and cytoplasmic mislocalisation of p27kip1, which in turn caused actin remodelling and increased migration in AGSGR cells.
MATERIALS AND METHODS: miRNA PCR arrays were used to identify changes in miRNA expression following G17 treatment of human gastric adenocarcinoma cells stably transfected with CCK2R (AGSGR). miR-222 was further investigated using primer assays and samples from hypergastrinemic mice and humans. Chemically synthesised mimics and inhibitors were used to assess cellular phenotypical changes associated with miR-222 dysregulation.
CONCLUSIONS: These data indicate a novel mechanism contributing to gastrin-associated gastric tumor development. miR-222 may also be a promising biomarker for monitoring gastrin induced premalignant changes in the stomach.

BACKGROUND: The increasing availability of resequencing data has led to a better understanding of the most important genes in cancer development. Nevertheless, the mutational landscape of many tumor types is heterogeneous and encompasses a long tail of potential driver genes that are systematically excluded by currently available methods due to the low frequency of their mutations. We developed LowMACA (Low frequency Mutations Analysis via Consensus Alignment), a method that combines the mutations of various proteins sharing the same functional domains to identify conserved residues that harbor clustered mutations in multiple sequence alignments. LowMACA is designed to visualize and statistically assess potential driver genes through the identification of their mutational hotspots.
RESULTS: We analyzed the Ras superfamily exploiting the known driver mutations of the trio K-N-HRAS, identifying new putative driver mutations and genes belonging to less known members of the Rho, Rab and Rheb subfamilies. Furthermore, we applied the same concept to a list of known and candidate driver genes, and observed that low confidence genes show similar patterns of mutation compared to high confidence genes of the same protein family.
CONCLUSIONS: LowMACA is a software for the identification of gain-of-function mutations in putative oncogenic families, increasing the amount of information on functional domains and their possible role in cancer. In this context LowMACA emphasizes the role of genes mutated at low frequency otherwise undetectable by classical single gene analysis. LowMACA is an R package available at http://www.bioconductor.org/packages/release/bioc/html/LowMACA.html. It is also available as a GUI standalone downloadable at: https://cgsb.genomics.iit.it/wiki/projects/LowMACA.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

PURPOSE: To evaluate the efficacy of adding lapatinib to capecitabine and oxaliplatin (CapeOx) in patients with previously untreated human epidermal growth factor receptor 2 (HER2) -amplified advanced gastroesophageal adenocarcinoma.
PATIENTS AND METHODS: Patients with HER2-positive advanced gastroesophageal adenocarcinoma were randomly assigned at a one-to-one ratio to CapeOx plus lapatinib 1,250 mg or placebo daily. Primary end point was overall survival (OS) in patients with centrally confirmed HER2 amplification in the primary efficacy population.
RESULTS: A total of 545 patients were randomly assigned, and 487 patients comprised the primary efficacy population. Median OS in the lapatinib and placebo arms was 12.2 (95% CI, 10.6 to 14.2) and 10.5 months (95% CI, 9.0 to 11.3), respectively, which was not significantly different (hazard ratio, 0.91; 95% CI, 0.73 to 1.12). Median progression-free survival in the lapatinib and placebo arms was 6.0 (95% CI, 5.6 to 7.0) and 5.4 months (95% CI, 4.4 to 5.7), respectively (hazard ratio, 0.82; 95% CI, 0.68 to 1.00; P = .0381). Response rate was significantly higher in the lapatinib arm: 53% (95% CI, 46.4 to 58.8) compared with 39% (95% CI, 32.9 to 45.3) in the placebo arm (P = .0031). Preplanned exploratory subgroup analyses showed OS in the lapatinib arm was prolonged in Asian and younger patients. No correlation was observed between HER2 immunohistochemistry status and survival. There were increased toxicities in the lapatinib arm, particularly diarrhea.
CONCLUSION: Addition of lapatinib to CapeOx did not increase OS in patients with HER2-amplified gastroesophageal adenocarcinoma. There were clear differences in the effect of lapatinib depending on region and age. Future studies could examine this correlation.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

BACKGROUND: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is thought to be a genetic disease caused by de novo mutations, though causative mutations have yet to be identified. We searched for de novo coding mutations among a carefully-diagnosed and clinically homogeneous cohort of 35 ROHHAD patients.
METHODS: We sequenced the exomes of seven ROHHAD trios, plus tumours from four of these patients and the unaffected monozygotic (MZ) twin of one (discovery cohort), to identify constitutional and somatic de novo sequence variants. We further analyzed this exome data to search for candidate genes under autosomal dominant and recessive models, and to identify structural variations. Candidate genes were tested by exome or Sanger sequencing in a replication cohort of 28 ROHHAD singletons.
RESULTS: The analysis of the trio-based exomes found 13 de novo variants. However, no two patients had de novo variants in the same gene, and additional patient exomes and mutation analysis in the replication cohort did not provide strong genetic evidence to implicate any of these sequence variants in ROHHAD. Somatic comparisons revealed no coding differences between any blood and tumour samples, or between the two discordant MZ twins. Neither autosomal dominant nor recessive analysis yielded candidate genes for ROHHAD, and we did not identify any potentially causative structural variations.
CONCLUSIONS: Clinical exome sequencing is highly unlikely to be a useful diagnostic test in patients with true ROHHAD. As ROHHAD has a high risk for fatality if not properly managed, it remains imperative to expand the search for non-exomic genetic risk factors, as well as to investigate other possible mechanisms of disease. In so doing, we will be able to confirm objectively the ROHHAD diagnosis and to contribute to our understanding of obesity, respiratory control, hypothalamic function, and autonomic regulation.