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Platinum therapy represents the first line of treatment in many malignancies but its high systemic toxicity limits the therapeutic dosage. Herein, we report the synthesis of carboplatin-like complexes with azide and alkyne functional groups and the formation of a platinum (II)-nuclear localization sequence peptide (Pt-NLS) hybrid to improve the import of platinum (II) complexes directly into the cell’s nucleus. The Pt-NLS hybrid successfully enters cells and their nuclei, forming Pt-induced nuclear lesions. The in vitro efficacy of Pt-NLS is high, superior to native carboplatin at the same concentration. The methodology used is simple and cost-effective and most importantly can easily be extended to load the Pt (II) onto other supports, opening new possibilities for enhanced delivery of Pt (II) therapy.

INTRODUCTION:Women with endometrial cancer should be screened for Lynch syndrome (LS), a hereditary cancer syndrome caused by germline mutations in DNA mismatch repair (MMR) genes. This information directly affects immediate clinical management including future risk reducing/surveillance strategies

Retrospective studies have demonstrated that nearly 50% of patients with ovarian cancer with normal cancer antigen 125 (CA125) levels have persistent disease; however, prospectively distinguishing between patients is currently impossible. Here, we demonstrate that for one patient, with the first reported fibroblast growth factor receptor 2 (FGFR2) fusion transcript in ovarian cancer, circulating tumor DNA (ctDNA) is a more sensitive and specific biomarker than CA125, and it can also inform on a candidate therapeutic. For a 4-year period, during which the patient underwent primary debulking surgery and chemotherapy, tumor recurrences, and multiple chemotherapeutic regimens, blood samples were longitudinally collected and stored. Whereas postsurgical CA125 levels were elevated only three times for 28 measurements, the FGFR2 fusion ctDNA biomarker was readily detectable by quantitative real-time reverse transcription-polymerase chain reaction (PCR) in all of these same blood samples and in the tumor recurrences. Given the persistence of the FGFR2 fusion, we treated tumor cells derived from this patient and others with the FGFR2 inhibitor BGJ398. Only tumor cells derived from this patient were sensitive to FGFR2 inhibitor treatment. Using the same methodologic approach, we demonstrate in a second patient with a different fusion that PCR and agarose gel electrophoresis can also be used to identify tumor-specific DNA in the circulation. Taken together, we demonstrate that a relatively inexpensive, PCR-based ctDNA surveillance assay can outperform CA125 in identifying occult disease.

Epithelial ovarian cancer (OvCa) is the most lethal female reproductive tract malignancy. A major clinical hurdle in patient management and treatment is that when using current surveillance technologies 80% of patients will be clinically diagnosed as having had a complete clinical response to primary therapy. In fact, the majority of women nonetheless develop disease recurrence within 18 mo. Thus, without more accurate surveillance protocols, the diagnostic question regarding OvCa recurrence remains framed as “when” rather than “if.” With this background, we describe the case of a 61-yr-old female who presented with a 3-mo history of unexplained whole-body rash, which unexpectedly led to a diagnosis of and her treatment for OvCa. The rash resolved immediately following debulking surgery. Nearly 1 yr later, however, the rash reappeared, prompting the prospect of tumor recurrence and requirement for additional chemotherapy. To investigate this possibility, we undertook a genomics-based tumor surveillance approach using a targeted 56-gene NGS panel and biobanked tumor samples to develop personalized ctDNA biomarkers. Although tumor-specific TP53 and PTEN mutations were detectable in all originally collected tumor samples, pelvic washes, and blood samples, they were not detectable in any biosample collected beyond the first month of treatment. No additional chemotherapy was given. The rash spontaneously resolved. Now, 2 yr beyond the patient's original surgery, and in the face of continued negative ctDNA findings, the patient remains with no evidence of disease. As this single case report suggests, we believe for the first time that ctDNA can provide an additional layer of information to avoid overtreatment.

Infantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. In eight of the families, we identified one of two disease-causing mutations, c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys), in PDGFRB. Intriguingly, one family did not have either of these PDGFRB mutations but all affected individuals had a c.4556T>C (p.Leu1519Pro) mutation in NOTCH3. Our studies suggest that mutations in PDGFRB are a cause of IM and highlight NOTCH3 as a candidate gene. Further studies of the crosstalk between PDGFRB and NOTCH pathways may offer new opportunities to identify mutations in other genes that result in IM and is a necessary first step toward understanding the mechanisms of both tumor growth and regression and its targeted treatment.

Background Endometrial cancer is the most common gynecologic malignancy, and its incidence and associated mortality are increasing. Despite the immediate need to detect these cancers at an earlier stage, there is no effective screening methodology or protocol for endometrial cancer. The comprehensive, genomics-based analysis of endometrial cancer by The Cancer Genome Atlas (TCGA) revealed many of the molecular defects that define this cancer. Based on these cancer genome results, and in a prospective study, we hypothesized that the use of ultra-deep, targeted gene sequencing could detect somatic mutations in uterine lavage fluid obtained from women undergoing hysteroscopy as a means of molecular screening and diagnosis. Methods and Findings Uterine lavage and paired blood samples were collected and analyzed from 107 consecutive patients who were undergoing hysteroscopy and curettage for diagnostic evaluation from this single-institution study. The lavage fluid was separated into cellular and acellular fractions by centrifugation. Cellular and cell-free DNA (cfDNA) were isolated from each lavage. Two targeted next-generation sequencing (NGS) gene panels, one composed of 56 genes and the other of 12 genes, were used for ultra-deep sequencing. To rule out potential NGS-based errors, orthogonal mutation validation was performed using digital PCR and Sanger sequencing. Seven patients were diagnosed with endometrial cancer based on classic histopathologic analysis. Six of these patients had stage IA cancer, and one of these cancers was only detectable as a microscopic focus within a polyp. All seven patients were found to have significant cancer-associated gene mutations in both cell pellet and cfDNA fractions. In the four patients in whom adequate tumor sample was available, all tumor mutations above a specific allele fraction were present in the uterine lavage DNA samples. Mutations originally only detected in lavage fluid fractions were later confirmed to be present in tumor but at allele fractions significantly less than 1%. Of the remaining 95 patients diagnosed with benign or non-cancer pathology, 44 had no significant cancer mutations detected. Intriguingly, 51 patients without histopathologic evidence of cancer had relatively high allele fraction (1.0%–30.4%), cancer-associated mutations. Participants with detected driver and potential driver mutations were significantly older (mean age mutated = 57.96, 95% confidence interval [CI]: 3.30–∞, mean age no mutations = 50.35; p-value = 0.002; Benjamini-Hochberg [BH] adjusted p-value = 0.015) and more likely to be post-menopausal (p-value = 0.004; BH-adjusted p-value = 0.015) than those without these mutations. No associations were detected between mutation status and race/ethnicity, body mass index, diabetes, parity, and smoking status. Long-term follow-up was not presently available in this prospective study for those women without histopathologic evidence of cancer. Conclusions Using ultra-deep NGS, we identified somatic mutations in DNA extracted both from cell pellets and a never previously reported cfDNA fraction from the uterine lavage. Using our targeted sequencing approach, endometrial driver mutations were identified in all seven women who received a cancer diagnosis based on classic histopathology of tissue curettage obtained at the time of hysteroscopy. In addition, relatively high allele fraction driver mutations were identified in the lavage fluid of approximately half of the women without a cancer diagnosis. Increasing age and post-menopausal status were associated with the presence of these cancer-associated mutations, suggesting the prevalent existence of a premalignant landscape in women without clinical evidence of cancer. Given that a uterine lavage can be easily and quickly performed even outside of the operating room and in a physician’s office-based setting, our findings suggest the future possibility of this approach for screening women for the earliest stages of endometrial cancer. However, our findings suggest that further insight into development of cancer or its interruption are needed before translation to the clinic., Navya Nair and colleagues describe a screening method for early identification of endometrial cancer. This method also reveals cancer mutations in women without cancer., Author Summary Why Was This Study Done? Endometrial cancer is the most common gynecologic malignancy in the United States. When detected early, endometrial cancer survival rates are improved. There are no screening methods that can detect either pre-malignant lesions or early-stage cancers. What Did the Researchers Do and Find? We conducted a prospective study in 107 women where uterine lavage fluid was analyzed for genetic mutations using ultra-deep, panel-based next-generation sequencing (NGS). Seven of the 107 women were identified by gold-standard histopathology as having endometrial cancer. All seven, even those with microscopic cancer foci, had significant cancer-associated gene mutations detected in their lavage fluid. Fifty-one women without histopathologic evidence of cancer had high allele fraction cancer-associated mutations. What Do These Findings Mean? This study provides the first evidence for the ability of an NGS-based approach to prospectively detect early-stage, microscopic endometrial cancer. Our study identified a previously unknown but highly prevalent landscape of driver and potential driver mutations in women who did not have histopathologic evidence of endometrial cancer. These findings suggest that while NGS-based analysis of uterine lavage can achieve the necessary sensitivity for endometrial cancer screening, further insights into the steps leading to endometrial cancer development and/or its interruption are still needed before this goal can be achieved. These findings, based on ultra-deep sequencing, may also have implications for understanding the development and clonal expansion of somatic cancer-driver mutations in apparently non-diseased tissues.

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21–22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.

Purpose: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC).Experimental Design: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided.Results: LOH was present in 59% of samples in a cell type–specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased ∼5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation.Conclusion: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.

BACKGROUND:High-grade serous ovarian and endometrial cancers are the most lethal female reproductive tract malignancies worldwide. In part, failure to treat these two aggressive cancers successfully centers on the fact that while the majority of patients are diagnosed based on current surveillance strategies as having a complete clinical response to their primary therapy, nearly half will develop disease recurrence within 18 months and the majority will die from disease recurrence within 5 years. Moreover, no currently used biomarkers or imaging studies can predict outcome following initial treatment. Circulating tumor DNA (ctDNA) represents a theoretically powerful biomarker for detecting otherwise occult disease. We therefore explored the use of personalized ctDNA markers as both a surveillance and prognostic biomarker in gynecologic cancers and compared this to current FDA-approved surveillance tools. METHODS AND FINDINGS:Tumor and serum samples were collected at time of surgery and then throughout treatment course for 44 patients with gynecologic cancers, representing 22 ovarian cancer cases, 17 uterine cancer cases, one peritoneal, three fallopian tube, and one patient with synchronous fallopian tube and uterine cancer. Patient/tumor-specific mutations were identified using whole-exome and targeted gene sequencing and ctDNA levels quantified using droplet digital PCR. CtDNA was detected in 93.8% of patients for whom probes were designed and levels were highly correlated with CA-125 serum and computed tomography (CT) scanning results. In six patients, ctDNA detected the presence of cancer even when CT scanning was negative and, on average, had a predictive lead time of seven months over CT imaging. Most notably, undetectable levels of ctDNA at six months following initial treatment was associated with markedly improved progression free and overall survival. CONCLUSIONS:Detection of residual disease in gynecologic, and indeed all cancers, represents a diagnostic dilemma and a potential critical inflection point in precision medicine. This study suggests that the use of personalized ctDNA biomarkers in gynecologic cancers can identify the presence of residual tumor while also more dynamically predicting response to treatment relative to currently used serum and imaging studies. Of particular interest, ctDNA was an independent predictor of survival in patients with ovarian and endometrial cancers. Earlier recognition of disease persistence and/or recurrence and the ability to stratify into better and worse outcome groups through ctDNA surveillance may open the window for improved survival and quality and life in these cancers.

Mouse chimeras from embryonic stem cells in which the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene had been targeted were crossed with normal females. First-generation (F(1)) G6PD(+/-) heterozygotes born from this cross were essentially normal; analysis of their tissues demonstrated strong selection for cells with the targeted G6PD allele on the inactive X chromosome. When these F(1) G6PD(+/-) females were bred to normal males, only normal G6PD mice were born, because: (i) hemizygous G6PD(-) male embryos died by E10.5 and their development was arrested from E7.5, the time of onset of blood circulation; (ii) heterozygous G6PD(+/-) females showed abnormalities from E8.5, and died by E11.5; and (iii) severe pathological changes were present in the placenta of both G6PD(-) and G6PD(+/-) embryos. Thus, G6PD is not indispensable for early embryo development; however, severe G6PD deficiency in the extraembryonic tissues (consequent on selective inactivation of the normal paternal G6PD allele) impairs the development of the placenta and causes death of the embryo. Most importantly, G6PD is indispensable for survival when the embryo is exposed to oxygen through its blood supply.

Bethlem myopathy is an early-onset benign myopathy characterized by proximal muscular weakness and multiple flexion contractures. It is a dominantly inherited disorder associated with mutations in the three COL6 genes encoding type VI collagen. We detected a ga substitution at +1 position of COL6A1 intron 3 in a four-generation Italian family affected by a mild form of Bethlem myopathy. The mutation results in the activation of a cryptic splice donor site at the 3′ end of exon 3, leading to the loss of 66 nucleotides and an “in-frame” deletion of 22 amino acids in the NH2-domain. Molecular analysis on fibroblasts of the propositus showed that the mutated mRNA was present and stable, but the mutated protein could not be detected. Western blot and immunofluorescence analyses showed a decreased level of collagen VI synthesis and deposition in fibroblasts of the propositus. Together, the results suggest that the mutated protein was highly unstable and rapidly degraded, and that the mild phenotype was caused by a reduced amount of normal collagen VI microfibrils. In addition, we demonstrated that lymphocytes can be used for the first mutation screening analysis of patients with Bethlem myopathy. © 2002 Wiley Periodicals, Inc. Muscle Nerve 25: 000–000, 2002

Targeted sequencing of cell-free (cfDNA) and circulating tumor DNA (ctcDNA) from blood enables detection of cancer-related mutations using minimally-invasive sample collection methods, and may make early detection of cancer possible, as well as improve monitoring of disease burden in translational research studies. We have developed a series of targeted panels for detection of multiple cancer-related mutations. The panels are designed to efficiently amplify damaged or short fragments of DNA derived from FFPE and cfDNA/ctcDNA, where hundreds of primer pairs can be amplified in a single tube from overlapping targets using only 10 ng input material, making these panels ideal for limiting liquid biopsy samples. A panel which covers known “hotspot” mutations in 56 oncology-related genes has been used in a pilot research study to monitor gynecological cancer in 11 women in a longitudinal study, which found a correlation between the presence of cancer mutations and morbidity and mortality. In 2 of 11 women, the initial absence of mutations above 1% allele-frequency was followed by the appearance of mutations in 1-3 genes at allele frequencies of 5-78% in the later time point. These 2 patients experienced increased morbidity or mortality. In 9 of the 11 women, no mutations were observed, and 6 remain in remission, while 3 are living with cancer. In an effort to further improve both workflow and performance, we are developing two technologies to incorporate into the panel design for future studies. The first will normalize library yield during PCR amplification for simple library pooling, which eliminates the requirement for library quantification and minimizes the time from sample to sequence. The second technology is a molecular ID (MID) system to tag each amplicon uniquely to allow data tracking to individual DNA fragments from the sample, and to increase confidence in variant calling by filtering PCR and sequencing errors. By incorporating technologies that reduce steps in the workflow, the likelihood of error is minimized, and combined with methods that increase confidence in low frequency variant calling, an ideal workflow for liquid biopsy samples is created. Citation Format: Jonathan C. Irish, Cassie A. Schumacher, Navya Nair, Olga Camacho-Vanegas, Jordan Rose- Figura, Ashley Wood, Sukhinder Sandhu, Sushma Chaluvadi, Sergey Chupreta, Laurie Kurihara, Timothy Harkins, John A. Martignetti, Vladimir Makarov. Targeted next-generation sequencing of cell-free tumor DNA to longitudinally monitor cancer burden and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5392. doi:10.1158/1538-7445.AM2017-5392

Tumor heterogeneity creates significant challenges for the generation and monitoring of treatment strategies. Circulating, cell-free DNA (cfDNA) can facilitate these processes by providing a noninvasive method to detect and track mutations without the need for multiple biopsies. To study both tumor heterogeneity and its relationship to cfDNA, we used samples taken from multiple positions on two metastatic tumors from a stage 3B ovarian carcinosarcoma patient as well as a cfDNA sample that was collected at the time of surgery. To maximize low frequency variant detection from next-generation sequencing (NGS) data, molecular identifiers (MIDs) were employed, allowing accurate distinction of true variants from PCR-induced errors along with sequencing artifacts. The use of MIDs to uniquely label input DNA generates tagged library molecules for the detection and removal of PCR duplicates while simultaneously preserving fragmentation and strand duplicates. By over-sequencing the MID tagged library, sequencing reads from PCR duplicates can be grouped based on their shared MID tag, generating a consensus sequence to identify and subsequently remove errors. To validate this technology, we performed low frequency spike-in experiments to 0.5% and 1% by combining Coriell NA12878 and HG005 genomic DNA as well as different cfDNA samples. Libraries were sequenced to 8000x coverage and a consensus sequence was generated with BMFtools (ARUP labs). All known variants present at 1% and 0.5% allele frequencies were maintained in the resulting data set. Further, true variants were preserved while sequencing and PCR-induced errors were removed, demonstrating improved sensitivity but also improved specificity using MIDs. After validation of the technology, libraries with MIDs were prepared from the ovarian carcinosarcoma FFPE and cfDNA samples. Libraries were sequenced on an Illumina® HiSeq® to a minimum of 13,000x coverage, and we determined data retention after de-duplication with and without the use of MIDs. We observed an increase in data retention for both Covaris-sheared FFPE and cfDNA libraries that led to a 2 to 3-fold increase in coverage using MIDs. Variant calling depicted significant inter-tumor heterogeneity in these samples as well as low frequency variants that represent intra-tumor heterogeneity. Specifically, we identified a pathogenic TP53 mutation present in one tumor sample and the cfDNA but absent from the second tumor. Additionally, we observed a significant loss of heterozygosity for only one of the tumors. We also identified low frequency, pathogenic mutations (as low as 1%) that are either unique to one tumor or unique to one sample from a tumor. This study highlights the use of MID technology to enable low frequency variant calling during disease diagnosis and tracking to facilitate precise, personalized treatment of complex cancers. Citation Format: Ashley Wood, Sukhinder Sandhu, Matthew Dashkoff, Olga Camacho-Vanegas, Laurie Kurihara, Timothy Harkins, John Martignetti, Vladimir Makarov, Peter Dottino. Characterizing tumorigenesis of ovarian cancer across metastatic tumors and circulating, cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-276. doi:10.1158/1538-7445.AM2017-LB-276

The statin family of cholesterol-lowering drugs is known to have pleiotropic properties which include anti-inflammatory and immunomodulatory effects. Statins exert their pleiotropic effects by altering expression of human immune regulators including pro-inflammatory cytokines. Previously we found that statins modulate virulence phenotypes of the human pathogen Pseudomonas aeruginosa, and sought to investigate if simvastatin could alter the host response to this organism in lung epithelial cells. Simvastatin increased the expression of the P. aeruginosa target genes KLF2, KLF6, IL-8 and CCL20. Furthermore, both simvastatin and P. aeruginosa induced alternative splicing of KLF6. The novel effect of simvastatin on wtKLF6 expression was found to be responsible for induction of the KLF6 regulated genes CCL20 and iNOS. Simvastatin also increased the adhesion of P. aeruginosa to host cells, without altering invasion or cytotoxicity. This study demonstrated that simvastatin had several novel effects on the pulmonary cellular immune response.

The genes coding for the ribosomal proteins (rp genes) L14 and L1 in the toad Xenopus laevis are contacted in the first exon by the frog protein, FIII/YY1, homolog of the human zinc-finger protein YY1, acting as repressor, activator and initiator of transcription. To investigate the functional significance of FIII/YY1 in the context of the two rp genes, the L14 region at nucleotide positions -105 to +44, including all of the first exon was linked to the chloramphenicol acetyltransferase (CAT) reporter gene; constructs with wild-type and mutated sites for FIII/YY1 were injected into nuclei of stage V-VI oocytes and analyzed for CAT activity. The same procedure was followed for constructs made with L1 sequences at nucleotide positions -17 to +1567. Mutations in the sites for FIII/YY1 did not change reporter activity, nor did overexpression of FIII/YY1 in the oocytes prior to injection with L1 and L14 constructs. Since oocytes are non-dividing cells, transfections were made of Xenopus kidney cells in culture with the same constructs and the results obtained in oocytes confirmed.

Obesity is a major public health concern, and complementary research strategies have been directed toward the identification of the underlying causative gene mutations that affect the normal pathways and networks that regulate energy balance. Here, we describe an autosomal-recessive morbid-obesity syndrome and identify the disease-causing gene defect. The average body mass index of affected family members was 48.7 (range = 36.7–61.0), and all had features of the metabolic syndrome. Homozygosity mapping localized the disease locus to a region in 3q29; we designated this region the morbid obesity 1 (MO1) locus. Sequence analysis identified a homozygous nonsense mutation in CEP19, the gene encoding the ciliary protein CEP19, in all affected family members. CEP19 is highly conserved in vertebrates and invertebrates, is expressed in multiple tissues, and localizes to the centrosome and primary cilia. Homozygous Cep19-knockout mice were morbidly obese, hyperphagic, glucose intolerant, and insulin resistant. Thus, loss of the ciliary protein CEP19 in humans and mice causes morbid obesity and defines a target for investigating the molecular pathogenesis of this disease and potential treatments for obesity and malnutrition.

In Xenopus laevis, as well as in other vertebrates, ribosomal proteins (r-proteins) are coded by a class of genes that share some organizational and structural features. One of these, also common to genes coding for other proteins involved in the translation apparatus synthesis and function, is the presence within their introns of sequences coding for small nucleolar RNAs. Another feature is the presence of common structures, mainly in the regions surrounding the 5′ ends, involved in their coregulated expression. This is attained at various regulatory levels: transcriptional, posttranscriptional, and translational. Particular attention is given here to regulation at the translational level, which has been studied during Xenopus oogenesis and embryogenesis and also during nutritional changes of Xenopus cultured cells. This regulation, which responds to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA (ribosomal protein mRNA) engaged on polysomes. A typical 5′ untranslated region characterizing all vertebrate rp-mRNAs analyzed to date is responsible for this translational behaviour: it is always short and starts with an 8–12 nucleotide polypyrimidine tract. This region binds in vitro some proteins that can represent putative trans-acting factors for this translational regulation.Key words: ribosomal proteins, snoRNA, translational regulation, Xenopus laevis.

Background Glutathione peroxidase 3 (GPX3) is a selenocysteine-containing antioxidant enzyme that reacts with hydrogen peroxide and soluble fatty acid hydroperoxides, thereby helping to maintain redox balance within cells. Serum levels of GPX3 have been found to be reduced in various cancers including prostrate, thyroid, colorectal, breast and gastric cancers. Intriguingly, GPX3 has been reported to be upregulated in clear cell ovarian cancer tissues and thus may have implications in chemotherapeutic resistance. Since clear cell and serous subtypes of ovarian cancer represent two distinct disease entities, the aim of this study was to determine GPX3 levels in serous ovarian cancer patients and establish its potential as a biomarker for detection and/or surveillance of papillary serous ovarian cancer, the most frequent form of ovarian tumors in women. Patients and Methods Serum was obtained from 66 patients (median age: 62 years, range: 22-89) prior to surgery and 65 controls with a comparable age-range (median age: 53 years, range: 25-83). ELISA was used to determine the levels of serum GPX3. The Mann Whitney U test was performed to determine statistical significance between the levels of serum GPX3 in patients and controls. Results Serum levels of GPX3 were found to be significantly lower in patients than controls (p = 1 × 10-2). Furthermore, this was found to be dependent on the stage of disease. While levels in early stage (I/II) patients showed no significant difference when compared to controls, there was a significant reduction in late stage (III/IV, p = 9 × 10-4) and recurrent (p = 1 × 10-2) patients. There was a statistically significant reduction in levels of GPX3 between early and late stage (p = 5 × 10-4) as well as early and recurrent (p = 1 × 10-2) patients. Comparison of women and controls stratified to include only women at or above 50 years of age shows that the same trends were maintained and the differences became more statistically significant. Conclusions Serum GPX3 levels are decreased in women with papillary serous ovarian cancer in a stage-dependent manner and also decreased in women with disease recurrence. Whether this decrease represents a general feature in response to the disease or a link to the progression of the cancer is unknown. Understanding this relationship may have clinical and therapeutic consequences for women with papillary serous adenocarcinoma.

The Krüppel-like transcription factor (KLF6) gene is a tumor suppressor gene (TSG) reported to be dysregulated and inactivated through loss of heterozygosity (LOH) and/or somatic mutation in a number of major human cancers. The aim of the present study was to examine KLF6 gene status and expression in head and neck squamous cell carcinomas (HNSCC). A collection of 81 well-characterized oral and oropharyngeal HNSCC samples were analyzed for evidence of KLF6 LOH and mutation and differences in expression patterns between normal and cancerous tissues and these findings were correlated with clinicopathological variables. We also tested the effect of KLF6 inhibition in HNSCC cell lines on proliferation and p21 expression. LOH was found in approximately 30% of cases and was strongly correlated with cancer progression, tumor recurrence and decreased patient survival. Overall, median survival of patients with LOH was less than half (19 vs. 41 months, p=0.036, stratified on stage) than those without loss. Risk of death for patients with LOH was 8 times greater independent of tumor size, nodal status, tobacco smoking or treatment modality (HR 7.89, 95% CI: 1.9-32.4). Subsequent analyses revealed KLF6 mutations in only 2 of 20 samples, but altered subcellular protein localization in 64% of tumors. Targeted stable reduction of KLF6 in HNSCC cell lines increased cellular proliferation while decreasing p21 expression. Taken together, these findings suggest that KLF6 LOH represents a clinically-relevant biomarker predicting patient survival and tumor recurrence and that dysregulation of KLF6 function plays an important role in HNSCC progression.The Krüppel-like transcription factor (KLF6) gene is a tumor suppressor gene (TSG) reported to be dysregulated and inactivated through loss of heterozygosity (LOH) and/or somatic mutation in a number of major human cancers. The aim of the present study was to examine KLF6 gene status and expression in head and neck squamous cell carcinomas (HNSCC). A collection of 81 well-characterized oral and oropharyngeal HNSCC samples were analyzed for evidence of KLF6 LOH and mutation and differences in expression patterns between normal and cancerous tissues and these findings were correlated with clinicopathological variables. We also tested the effect of KLF6 inhibition in HNSCC cell lines on proliferation and p21 expression. LOH was found in approximately 30% of cases and was strongly correlated with cancer progression, tumor recurrence and decreased patient survival. Overall, median survival of patients with LOH was less than half (19 vs. 41 months, p = 0.036, stratified on stage) than those without loss. Risk of death for patients with LOH was 8 times greater independent of tumor size, nodal status, tobacco smoking or treatment modality (HR 7.89, 95% CI: 1.9-32.4). Subsequent analyses revealed KLF6 mutations in only 2 of 20 samples, but altered subcellular protein localization in 64% of tumors. Targeted stable reduction of KLF6 in HNSCC cell lines increased cellular proliferation while decreasing p21 expression. Taken together, these findings suggest that KLF6 LOH represents a clinically-relevant biomarker predicting patient survival and tumor recurrence and that dysregulation of KLF6 function plays an important role in HNSCC progression.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor and possesses a high incidence of 10p loss. The KLF6 (Kruppel-like transcription factor) tumor suppressor gene on 10p15 is inactivated by loss of heterozygosity (LOH) and/or somatic mutation in a number of human cancers and forced expression of KLF6 in GBM lines inhibits their growth and transformation. In addition, increased expression of its alternatively spliced, cytoplasmic isoform KLF6-SV1 has now been shown to play a role in cancer pathogenesis. On the basis of these findings we examined the role of KLF6 and KLF6-SV1 in the development and progression of GBM. LOH analysis of 17 primary GBM patient samples using KLF6-specific microsatellite markers revealed that 88.2% (15/17) had LOH of the KLF6 locus. Interestingly, no KLF6 somatic mutations were identified. RNA analysis revealed concomitant decreases in all primary GBM tumors (n = 11) by approximately 80% in KLF6 expression (p < 0.001) coupled with increased KLF6-SV1 expression (p < 0.001) when compared to normal astrocytes. To determine the biological relevance of these findings, we examined the effect of KLF6 expression and KLF6-SV1 knockdown in A235 and CRL2020 cell lines. Reconstitution of KLF6 decreased cell proliferation by almost 50%, whereas targeted KLF6 reduction increased cell proliferation 2.5-4.5 fold. Conversely, targeted KLF6-SV1 reduction decreased cell proliferation by 50%. Taken together, our findings demonstrate that KLF6 allelic imbalance and decreased KLF6 and increased KLF6-SV1 expression are common findings in primary GBM tumors, and these changes have antagonistic effects on the regulation of cellular proliferation in GBM cell lines.

Gynecologic malignancies affect over 94,000 women in the U.S. and will result in close to 29,000 deaths this year alone. Currently available biomarkers are inadequate for the early detection of disease recurrence. The development of more sensitive and accurate biomarkers represents a critical and immediate necessity towards improving both patient outcome and quality of life. The measurement of circulating tumor DNA (ctDNA), the so-called “liquid biopsy,” represents a powerful emerging technology capable of providing accurate assessment of both tumor behavior and disease burden, and has great potential for revolutionizing our standard approach to tumor surveillance. The ability to translate the various ctDNA detection technologies from the laboratory to the clinic and begin defining clinical utility will in part be determined by the time and cost efficiencies of the pipelines and inherent detection sensitivities. We have developed a precision-medicine based approach that couples targeted tumor-specific mutation identification for each patient to droplet digital PCR-based (ddPCR) ctDNA detection. Using two rapid-turnaround sequencing panels (targeted full-gene PacBio single-molecule real time (SMRT) long-read sequencing coupled with the Ion AmpliSeq™Cancer Hotspot Panel v2) in lieu of more expensive and time-consuming approaches like whole-exome or whole-genome sequencing, we comprehensively identified tumor mutations for each of our ovarian and endometrial cancer patients by sequencing normal and tumor DNA from each patient. Analysis of an initial cohort of ovarian and endometrial cancer patients resulted in mutation identification frequencies comparable to previously reported data with mutations in TP53, PTEN, and PIK3CA being most frequently represented. Candidate mutations for ctDNA detection were identified for greater than 80% of subjects using this approach. By targeting specific genes, we raise the likelihood that identified tumor mutations are drivers essential to the disease, rather than passenger mutations that may be selected out during disease progression. ctDNA biomarkers specific to each patient's tumor mutations were generated and tested using ddPCR, allowing for detection down to 0.05% mutant allele fraction. ctDNA levels were assessed longitudinally from prospectively collected serums during multiple time points throughout each patient's clinical course and compared to clinical findings and current gold-standard serum and radiologic tests. ctDNA was successfully detected in all subjects; and to our knowledge for the first time in endometrial cancer. We demonstrate that our molecular approach allows for rapid, highly sensitive and accurate assessment of patient tumor status and, for some patients, is an improved and better predictor of outcome than current gold-standard surveillance strategies. Citation Format: Elena B. Pereira, Olga Camacho-Vanegas, Sanya Anand, Chanpreet Singh, Andrew Uzilov, Robert Sebra, David Chappell, Peter Dottino, John A. Martignetti. Precision molecular biomarkers for the surveillance of gynecologic malignancies: Rapid and efficient pipeline for the design and highly sensitive detection of circulating tumor DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2398. doi:10.1158/1538-7445.AM2015-2398

The tumor suppressor KLF6 is a member of the Kruppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P

SummaryA difference in the prevalence of venous thromboembolism (TE) in major human groups has been described and an uneven distribution of FV Leiden mutation over the world has recently been reported.We investigated FV Leiden mutation in 584 apparently healthy sub#jects mostly from populations different from those previously investi#gated: 170 Europeans (Spanish, Italians), 101 sub-saharan Africans (Fon, Bariba, Berba, Dendi), 115 Asians (Indonesians, Chinese, Tharus), 57 Amerindians (Cayapa), 84 Afroamericans (Rio Cayapa, Viche), and 57 Ethiopians (Amhara, Oromo).The mutation was detected in only 1/115 Asian (Tharu) and in 5/170 Europeans (4 Italians, 1 Spanish).These data confirm that in non-Europeans the prevalence of FV mutation is at least 7 times lower than in Europeans and provide indirect evidence of a low prevalence not only of the FV Leiden gene but also of other genes leading to more severe thrombophilia. Finally, findings from the literature together with those pertaining to this study clearly show a marked heterogeneity among Europeans.

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA–mediated KLF6 SV1 inhibition reduces growth by ∼50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.

A highly spread polymorphism flanking the 3 · Ca1 human IG heavy chain gene was identified. This polymorphism allowed the detection of an internal duplication within the 3ae flanking region of both Ca1 and Ca2. This region has a regulatory function with four enhancer structures also present at the 3ae end of the human Ca2 as well as in that of mouse and rat single Ca genes. The 5682-bp sequence of clone lpl8 described here starts 3ae of Ca1 and presents three open reading frames; one of them contains part of the tandem repeats with the 20-bp consensus described previously that is expressed in a poly(A)+ RNA and found in three dbEST clones of the human tonsillar cDNA library. Here, we demonstrate that in the CLF1 B lymphoblastoid cell line, this transcript is associated with polysomes. We also discuss the possibility of the presence of a new regulatory gene that does not encode an immunoglobulin and maps in the human IG heavy chain gene cluster. © 1998 Elsevier Science B.V. All rights reserved.

The hnRNP A1 transcript has a relatively short 5'- untranslated region (UTR) starting with a pyrimidine tract similar to that of mRNAs encoded by the TOP [terminal oligo(pyrimidine)] genes in vertebrates. Such genes code for ribosomal proteins and for other proteins directly or indirectly involved in the production and function of the translation apparatus. As expected from the role of the pyrimidine tract in the translational regulation of TOP mRNAs, the A1 mRNA is more efficiently loaded onto polysomes in growing than in resting cells. On the other hand, a less stringent regulation with respect to that of other TOP mRNAs is observed, partially due to the presence of multiple transcription start sites within the pyrimidine tract, where transcripts with shorter TOP sequences are less sensitive to regulation. Thus, from the point of view of structural features and translation behaviour the A1 mRNA can be included in the class of TOP genes, suggesting a possible role of A1 in translation. Interestingly, a TOP-like behaviour was observed for hnRNP I mRNA but not for hnRNP C1/C2 and A2/B1 mRNAs, indicating the existence of two classes of hnRNPs with different translational regulation.

Hereditary cancer syndromes represent a powerful and tractable biologic system for identifying cancer-causing genes. Though the syndromes themselves may be rare, their study can provide insights into the basis of the more common sporadic forms of the cancer. Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal dominant bone dysplasia / bone cancer syndrome. This hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, and pathologic fractures. Most notably, 35% of affected individuals develop bone MFH, a sarcoma that in its sporadic form accounts for 6% of all bone cancers and is believed to be etiologically related to osteosarcoma. Indeed, one of our affected family members developed histologically-proven osteosarcoma, thus further supporting a genetic link between these tumor types. Using a linkage based approach, we previously mapped the DMS-MFH tumor suppressor gene locus to chromosome 9p21-22 (1) and then through LOH analysis demonstrated that hereditary and sporadic forms of MFH most likely share a single underlying genetic etiology (2). We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously unrecognized terminal exons of the methylthioadenosine phosphorylase (MTAP) gene. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine/methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Intriguingly, two of the novel MTAP exons arose from early and independent retroviral integration events in primate genomes at least 40 MYR ago and since their genomic integration have gained a functional role. Six distinct retroviral-sequence containing MTAP isoforms, each of which can physically interact with archetype MTAP (i.e., exons 1-8), are identified. The disease-causing / cancer-associated mutations occur within one of these retroviral-derived exons. The mutations result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Based on these findings in a hereditary form of bone sarcoma, we then analyzed the expression of these MTAP isoforms in a sample set (n=16) of sporadic osteosarcoma samples. All tumor samples expressed similar levels of the archetype MTAP RNA sequence but the expression pattern of the splice variants varied markedly between nearly all the samples. The majority of samples did not express SV1 (n=11/16) and nearly half did not express SV6 (n=9/16). Taken together, these results identify the first gene involved in the development of bone MFH / osteosarcoma and have potential implications for the treatment of this human cancer. References: 1. Am J Hum Genet. 64:801-7; 1999. 2. Genes Chromosomes Cancer. 27:191-5; 2000. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-89. doi:1538-7445.AM2012-LB-89

This study suggests a macroheterogeneity in prevalence of platelet glycoprotein PL(A1/A2) polymorphism in different ethnic populations. In patients undergoing percutaneous transluminal coronary angioplasty, this polymorphism does not represent an independent risk factor but seems to be implicated in restenosis after percutaneous transluminal coronary angioplasty.

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription–PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A → G substitution at nucleotide −2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G → A substitution at nucleotide −1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far—the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

Frequency of Factor V (FV) Leiden and C677T Methylenetetrahydrofolate Reductase (MTHFR) Mutations in Colombians