Molecular alterations of endometrial and ovarian tumorigenesis in Lynch syndrome mutation carriers and the general population

Endometrial and ovarian cancers are among the most prevalent malignancies in females all around the world. Type I subset of these carcinomas exhibit many similarities in their genetic and epigenetic profiles. Lynch syndrome (LS) is one of the most prevalent hereditary cancer susceptibility syndromes in the world. LS is a result of defective mismatch repair (MMR) caused by a germline mutation in MMR genes, which combined with other molecular alterations, is known to accelerate tumorigenesis. In addition to a high prevalence in colon cancer, type I endometrial and ovarian cancers predominate in women with LS. Apart from the MMR abnormalities, the molecular profile of LS-associated ovarian cancer remains unknown. Moreover, the developmental changes occurring in LS patients and in the general population prior to endometrial and ovarian cancer are poorly understood. Type I endometrial and ovarian non-serous carcinomas are believed to originate from the endometrial lining of the uterus, termed the endometrium. Women with LS have been offered regular gynecological surveillance in Finland since 1996. This surveillance program provides invaluable consecutive endometrial samples before cancer diagnosis and represents an excellent model with which to investigate the molecular changes resulting in the development of endometrial and ovarian tumors. The aims of this thesis were to identify and compare the molecular alterations in LS-associated and sporadic ovarian cancer, and to determine genetic, epigenetic and gene expression alterations in consecutive specimens prior to the appearance of endometrial and ovarian cancer. In total, 213 endometrial and ovarian carcinomas, as well as endometrial biopsy specimens from 66 LS mutation carriers were compared to 197 sporadic specimens of the corresponding histological types and profiled with established genetic and partly novel epigenetic markers. Immunohistochemistry (IHC) was used to analyze expression of MMR, ARID1A, and L1CAM genes, whereas epigenetic DNA methylation alterations of 37 tumor suppressor genes (TSGs) were evaluated using both commercial and custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assays. Additionally, ovarian carcinomas were investigated by IHC for p53 protein, hypomethylation of LINE-1 marker (a retrotransposon) was detected by MS-MLPA and we also conducted a mutational analysis of hotspot sites in KRAS, BRAF, and PIK3CA by PCR, followed by sequencing. Novel molecular characteristics of LS-associated ovarian cancer were identified: An extremely high frequency of loss of ARID1A protein expression, MMR deficiency, no BRAF and KRAS mutations, normal p53 protein expression, a unique hypermethylation of selected TSGs, and an absence of LINE-1 hypomethylation in endometrioid and clear cell ovarian carcinomas, and frequent L1CAM overexpression specifically in clear cell ovarian cancer. Molecular analyses of LS surveillance specimens revealed closely related pathways in endometrial and ovarian type I tumorigenesis. For example, both MMR deficiency and TSG promoter methylation of specific genes appeared in histologically normal endometrial tissue preceding endometrial and ovarian cancer and there was ARID1A loss in complex hyperplasia with or without atypia prior to the appearance of the endometrial cancer. Additionally, we identified a high degree of similarity in the molecular alterations present in the hyperplastic lesions that occurred prior to or concurrently with the detection of endometrial or ovarian carcinoma collected from the same patient. This discovery suggests that endometrial hyperplasia may contribute to the development of ovarian tumors in addition to its well-established role in endometrial tumorigenesis. The findings provide novel and valuable information about the gynecological tumorigenesis of LS as well as the corresponding tumor with a sporadic origin. The results may facilitate the prediction of the malignant potential of pre-neoplastic specimens, guide treatment decisions and identify those women who could benefit from prophylactic surgery.Kohtu- ja munasarjasyöpä ovat naisten yleisimpiä syöpiä, kun taas Lynchin syndrooma on kaikkein yleisin perinnöllinen syöpäoireyhtymä maailmassa, aiheuttaen naiskantajilleen kohonneen riskin sairastua elinaikanaan tyypin 1 kohtu- (jopa 57 %) ja munasarjasyöpiin (jopa 24 %). Lynchin syndrooma on seurausta sukusoluissa tapahtuneesta mutaatiosta DNA:n emäspariutumisvirheitä korjaavissa (MMR) geeneissä, mikä aiheuttaa puutteita DNA:n korjauksessa ja lisää syöpäalttiutta eri kudoksissa. Lynchin syndroomaan liittyvät munasarjasyövät eroavat ilmiasultaan yleisesti väestössä esiintyvästä munasarjasyövästä, mutta MMR-virheiden ohella muita tekijöitä ei ole aiemmin tunnistettu. Myös kohtu- ja munasarjasyövän kehitykseen johtavat muutokset Lynchin syndrooman mutaationkantajilla sekä yleisesti väestössä tunnetaan heikosti. Työssä tutkimme erilaisia tunnettuja geneettisiä ja osin uusia epigeneettisiä markkereita, joiden toiminta usein muuttuu gynekologisissa syövissä. Geneettinen muutos tarkoittaa DNA-sekvenssin rakenteellista mutaatiota, kun taas epigeneettinen muutos aiheuttaa periytyviä muutoksia geenien toiminnassa, DNA-sekvenssin pysyessä ennallaan. Tutkimuksissa havaitsimme ainutlaatuisen molekyyliprofiilin Lynchin syndroomaan liittyvässä munasarjasyövässä. Tärkeimpiä löydöksiä olivat erittäin yleinen ARID1A:n valkuaistuotteen puutos, MMR-koneiston häiriöt, valikoitujen kasvunrajoitegeenien hypermetylaatio, sekä L1CAM-valkuaisen ylituotanto erityisesti kirkassoluisissa munasarjasyövissä. Nykytietämyksen valossa tyypin 1 kohtu- ja munasarjasyövän kaikkein aikaisimpien muutosten uskotaan näkyvän jo kohdun limakalvolla. Suomessa Lynchin syndrooman mutaationkantajanaiset ovat osallistuneet gynekologiseen seulontaan vuodesta 1996, jonka yhteydessä otetaan limakalvonäyte kohdusta. Seulonnan ansiosta pystymme tutkimaan kliinisiä näytteitä kohdun limakalvolta jo vuosia ennen potilaiden syöpädiagnoosia. Tutkimusta varten keräsimme yli 200 kohtu- ja munasarjanäytettä lähes 70 Lynchin syndrooman mutaationkantajalta, jotka ovat sairastuneet kohtu- ja/tai munasarjasyöpään ja/tai kohtusyövän esiasteeseen. Lisäksi keräsimme syöpä- ja esiastediagnooseja edeltävät potilaiden seulontanäytteet yli kahdenkymmenen vuoden ajalta koko Suomen laajuisesti. Tämä keräys on hyvin ainutlaatuinen, eikä vastaavaa ole tietääksemme tehty aiemmin. Lynchin syndroomapotilaiden syöpä- ja seulontanäytteiden analyysit paljastivat läheisesti yhteen liittyviä kehitysreittejä tyypin 1 kohtu- ja munasarjasyövässä. MMR-puutokset ja valikoitujen geenien säätelyalueiden hypermetylaatio olivat havaittavissa jo histologisesti normaalilta kohdun limakalvolta jopa useita vuosia ennen kohtu- ja munasarjasyöpää, ja ARID1A:n valkuaistuote hävisi jo osassa kohtusyöpää edeltävissä kohtusyövän esiasteissa. Lisäksi, kohtu- ja munasarjasyöpää edeltävät esiasteet sisälsivät paljon samoja molekyylitason muutoksia kuin myöhemmin ilmenevät kasvaimet. Tämä mielenkiintoinen löydös viittaa siihen, että kohtusyövän esiasteet saattavat olla osallisina myös munasarjasyövän kehityksessä. Tutkimustyö tuo uutta ja tärkeää tietoa munasarja- ja kohtusyövän syntymekanismeista sekä kulusta Lynchin syndrooman mutaation kantajilla ja yleisesti väestössä ilmenevissä syövissä. Tutkimuksessa tehtyjä ainutlaatuisia löydöksiä voidaan mahdollisesti hyödyntää myöhemmin sekä perinnöllisen että satunnaisesti väestössä ilmenevän munasarja- ja kohtusyövän diagnostiikassa sekä potilaiden jakamisessa erityyppistä ja räätälöityä hoitoa vaativiin ryhmiin. Työn tulokset voivat edesauttaa erityisesti kohtusyöpää edeltävien esiasteiden pahanlaatuisuuden arvioinnissa, ohjata hoitopäätöksiä ja tunnistaa niitä potilaita, jotka hyötyvät lääkkeellisestä hoidosta tai ennaltaehkäisevästä kirurgiasta

Converging endometrial and ovarian tumorigenesis in Lynch syndrome : Shared origin of synchronous carcinomas

AbstractObjective The diagnosis of carcinoma in both the uterus and the ovary simultaneously is not uncommon and raises the question of synchronous primaries vs. metastatic disease. Targeted sequencing of sporadic synchronous endometrial and ovarian carcinomas has shown that such tumors are clonally related and thus represent metastatic disease from one site to the other. Our purpose was to investigate whether or not the same applies to Lynch syndrome (LS), in which synchronous cancers of the gynecological tract are twice as frequent as in sporadic cases, reflecting inherited defects in DNA mismatch repair (MMR). Methods MMR gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nationwide registry. Endometrial (n = 35) and ovarian carcinomas (n = 23), including 13 synchronous carcinoma pairs, were collected as well as endometrial hyperplasias (n = 56) and normal endometria (n = 99) from a surveillance program over two decades. All samples were studied for MMR status, ARID1A and L1CAM protein expression and tumor suppressor gene promoter methylation, and synchronous carcinomas additionally for somatic mutation profiles of 578 cancer-relevant genes. Results Synchronous carcinomas were molecularly concordant in all cases. Prior or concurrent complex (but not simple) endometrial hyperplasias showed a high degree of concordance with endometrial or ovarian carcinoma as the endpoint lesion. Conclusions Our investigation suggests shared origins for synchronous endometrial and ovarian carcinomas in LS, in analogy to sporadic cases. The similar degrees of concordance between complex hyperplasias and endometrial vs. ovarian carcinoma highlight converging pathways for endometrial and ovarian tumorigenesis overall.Peer reviewe

The Role of Chromosomal Instability and Epigenetics in Colorectal Cancers Lacking beta-Catenin/TCF Regulated Transcription

All colorectal cancer cell lines except RKO displayed active beta-catenin/TCF regulated transcription. This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines (with active Wnt signaling) and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various beta-catenin localizations as a surrogate marker for beta-catenin activation. The immunohistochemistry of selected genes (IFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors withmembranous beta-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.Peer reviewe

Faba bean protein gelling properties affected by wet fractionation processes with and without spray-drying

Previous studies have demonstrated that legume proteins have high potential to lose their native functional properties during conventional wet fractionation, since heating and drying in the processes can lead to protein aggregation and solubility reduction. However, the effect of this process on faba bean protein has not been investigated thoroughly before, according to the literature review. Therefore, the aim of this study was to investigate the influence of wet fractionation process with and without spray drying on faba bean protein gelling properties. The hypothesis was that protein isolate produced with isoelectric precipitation without spray drying would have better solubility and gelling properties than spray dried isolate. Faba bean protein concentrate (FBPC) was used to prepare faba bean protein isolate with alkaline extraction followed by isoelectric precipitation, which was then dispersed into water and neutralized (faba bean protein slurry, FBPS). Some of FBPS was spray-dried (pilot faba bean protein isolate, FBPI-P). Commercial faba bean protein isolate (FBPI-C) was used as a reference sample. Their (FBPC, FBPS, FBPI-P, FBPI-C) functionality was studied by measuring thermal denaturation (DSC), protein solubility, particle size and light microscope pictures. In addition, glucono delta lactone (GDL) induced oil-in-water emulsion gels were prepared from FBPS, FBPI-P and FBPI-C. Water holding capacity (WHC), rheological and texture properties were measured from the gel samples. Surprisingly the obtained results of faba bean in this study were different from previous research on other legume proteins. It was found that spray-dried FBPI-P showed better gelling properties than wet based FBPS. This might be due to differences in thermal denaturation. Based on DSC curves, FBPI-C was found to be fully denatured and in a poor solubility. Interestingly, GDL-induced gels had strongest gel properties, which might be highly related to industrial manufacturing process of FBPI-C. When considering the theoretical environmental impact of these processes, FBPS could show potential of being more environmentally friendly process, as it could be more energy efficient than processes which require drying and heating. It was demonstrated that FBPS showed almost as good functional properties as the pilot spray-dried isolate. Hence, FBPS could work as functional and more environmentally friendly option for food industry. Future study could be conducted in near future about FBPS safety, technological applications, process optimization and environmental impact

Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

<div><p>Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of <i>RSK4</i>, <i>SPARC</i>, and <i>HOXA9</i> were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of <i>RSK4</i>, <i>SPARC</i>, <i>PROM1</i>, <i>HOXA10</i>, <i>HOXA9</i>, <i>WT1-AS</i>, <i>SFRP2</i>, <i>SFRP5</i>, <i>OPCML</i>, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.</p></div

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder