One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain ...and infertility
, and are a common cause of hysterectomy
. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA2
. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex
, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice
. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.
Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome caused by germline fumarate hydratase (FH) mutations and characterized by uterine and cutaneous leiomyomas ...and renal cell cancer. Currently, there is no generally approved method to differentiate FH-deficient uterine leiomyomas from other leiomyomas. Here, we analyzed 3 antibodies (S-(2-succino)-cysteine 2SC, aldo-keto reductase family 1, member B10 AKR1B10, and FH) as potential biomarkers. The study consisted of 2 sample series. The first series included 155 formalin-fixed paraffin-embedded uterine leiomyomas, of which 90 were from HLRCC patients and 65 were sporadic. The second series included 1590 unselected fresh frozen leiomyomas. Twenty-seven tumors were from known HLRCC patients, while the FH status for the remaining 1563 tumors has been determined by copy number analysis and Sanger sequencing revealing 45 tumors with monoallelic (n=33) or biallelic (n=12) FH loss. Altogether 197 samples were included in immunohistochemical analyses: all 155 samples from series 1 and 42 available corresponding formalin-fixed paraffin-embedded samples from series 2 (15 tumors with monoallelic and 7 with biallelic FH loss, 20 with no FH deletion). Results show that 2SC performed best with 100% sensitivity and specificity. Scoring was straightforward with unambiguously positive or negative results. AKR1B10 identified most tumors accurately with 100% sensitivity and 99% specificity. FH was 100% specific but showed slightly reduced 91% sensitivity. Both FH and AKR1B10 displayed also intermediate staining intensities. We suggest that when patient's medical history and/or histopathologic tumor characteristics indicate potential FH-deficiency, the tumor's FH status is determined by 2SC staining. When aberrant staining is observed, the patient can be directed to genetic counseling and mutation screening.
Uterine leiomyomas, or fibroids, are very common smooth muscle tumors that arise from the myometrium. They can be divided into distinct molecular subtypes. We have previously shown that ...3′RNA‐sequencing is highly effective in classifying archival formalin‐fixed paraffin‐embedded (FFPE) leiomyomas according to the underlying mutation. In this study, we performed 3′RNA‐sequencing with 111 FFPE leiomyomas previously classified as negative for driver alterations in mediator complex subunit 12 (MED12), high mobility group AT‐hook 2 (HMGA2), and fumarate hydratase (FH) by Sanger sequencing and immunohistochemistry. This revealed 43 tumors that displayed expression features typically seen in HMGA2‐positive tumors, including overexpression of PLAG1. We explored 12 such leiomyomas by whole‐genome sequencing to identify their underlying genomic drivers and to evaluate the feasibility of detecting chromosomal driver alterations from FFPE material. Four tumors with significant HMGA2 overexpression at the protein‐level served as controls. We identified chromosomal rearrangements targeting either HMGA2, HMGA1, or PLAG1 in all 16 tumors, demonstrating that it is possible to detect chromosomal driver alterations in archival leiomyoma specimens as old as 18 years. Furthermore, two tumors displayed biallelic loss of DEPDC5 and one tumor harbored a COL4A5–COL4A6 deletion. These observations suggest that instead of only HMGA2‐positive leiomyomas, a distinct leiomyoma subtype is characterized by rearrangements targeting either HMGA2, HMGA1, or PLAG1. The results indicate that the frequency of HMGA2‐positive leiomyomas may be higher than estimated in previous studies where immunohistochemistry has been used. This study also demonstrates the feasibility of detecting chromosomal driver alterations from archival FFPE material.
Uterine leiomyomas, or fibroids, are very common smooth muscle tumors. Their potential to metastasize or transform into leiomyosarcomas is extremely low. Here, we report a patient who underwent ...hysterectomy due to a large leiomyoma and who was diagnosed with pulmonary tumors seven and nine years later. Histopathological re-evaluation confirmed the cellular leiomyoma diagnosis for the uterine tumor, whereas the pulmonary tumors met the diagnostic criteria of a leiomyosarcoma. Whole-exome sequencing revealed very similar mutational profiles in all three tumors, including a somatic homozygous deletion in a rare, but well-established leiomyoma driver gene FH. Tumor evolution analysis confirmed the clonal origin of all three tumors. In addition to mutations shared by all three tumors, pulmonary tumors harbored additional alterations affecting e.g. the cancer-associated genes NRG1 and MYOCD. The second pulmonary leiomyosarcoma harbored additional changes, including a mutation in FGFR1. In global gene expression profiling, the uterine tumor showed similar expression patterns as other FH-deficient leiomyomas. Taken together, this comprehensive molecular data supports the occasional metastatic capability and malignant transformation of uterine leiomyomas. Further studies are required to confirm whether FH-deficient tumors and/or tumors with cellular histopathology have higher malignant potential than other uterine leiomyomas.
Abstract
HLRCC is an autosomal dominant tumor predisposition syndrome characterized by cutaneous and uterine leiomyomas (ULs), and increased risk for aggressive type 2 papillary renal cell carcinoma ...(RCC). The syndrome is caused by germline mutations in fumarate hydratase (FH). Loss of FH causes dysfunction of the Crebs cycle and an increase of succinated proteins, which can be detected using anti-2-succinocysteine (2SC) antibody. Recently, we have observed that aldo-keto reductase family 1, member B10 (AKR1B10) is overexpressed in RNA-level in ULs with biallelic loss of FH. Here, we compared anti-AKR1B10, anti-2SC, and anti-FH antibodies in detection of FH loss in protein-level in HLRCC-associated ULs.
The study material consisted of 142 formalin-fixed paraffin-embedded (FFPE) UL samples collected at the University hospitals in Finland. The sample series include 77 FH-deficient UL samples from 25 HLRCC patients and 65 sporadic conventional ULs. HLRCC background was verified by detected germline mutation of FH. Four 0.8 mm cores of representative FFPE tumor tissue were used to construct tissue microarrays. Immunohistochemistry (IHC) was executed using an anti-AKR1B10 (1:300, H00057016-M01, Abnova), anti-2SC (1:2000, provided by Dr. Norma Frizzell), and anti-FH (1:1000, sc-100743, Santa Cruz Biotechnology, Inc) antibodies. Expression was detected by BrightVision (Immunologic) and DAB Quanto (Thermo Fisher Scientific) systems. Pathologist specialized in gynecological pathology evaluated the IHC staining from the smooth muscle tumor cells.
AKR1B10 and 2SC detected all 77 HLRCC samples. AKR1B10 displayed either mild (12/77, 16%) or strong (65/77, 84%) expression and succination level was strong in all cases. Loss of FH expression was displayed in 68/77 (88%) of the FH-deficient ULs. All conventional ULs displayed negative AKR1B10 and 2SC staining and expressed FH. FH was expressed either mildly (15/65, 23%) or strongly (50/65, 77%) in the samples.
Reliable methods for FH-deficient UL detection in the clinics are currently lacking. Here, comparison of AKR1B10, 2SC, and FH showed that all three biomarkers identified HLRCC-ULs with 100% specificity. The sensitivity was 100% with AKR1B10 and 2SC, and 88% with FH. It seems that even with known mutation in FH, in some cases the likely inactive protein is retained in tumor cells and is thus detectable by anti-FH.
To conclude, we show that anti-AKR1B10 and anti-2SC detect FH-deficient ULs with 100% specificity and sensitivity. Anti-FH is lacking sensitivity in 12% of HLRCC cases. Reliable detection of FH-deficient ULs enables the patients and their families to be directed to genetic counseling, family planning, and regular renal monitoring.
Citation Format: Terhi Ahvenainen, Outi Uimari, Anne Ahtikoski, Kati Kämpjärvi, Ralf Bützow, Pia Vahteristo. Comparison of AKR1B10, 2SC, and FH as biomarkers for HLRCC detection abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3992.
Meningiomas are the most common primary tumors of the CNS and account for up to 30% of all CNS tumors. An increased risk of meningiomas has been associated with certain tumor-susceptibility ...syndromes, especially neurofibromatosis type II, but no gene defects predisposing to isolated familial meningiomas have thus far been identified. Here, we report on a family of five meningioma-affected siblings, four of whom have multiple tumors. No NF2 mutations were identified in the germline or tumors. We combined genome-wide linkage analysis and exome sequencing, and we identified in suppressor of fused homolog (Drosophila), SUFU, a c.367C>T (p.Arg123Cys) mutation segregating with the meningiomas in the family. The variation was not present in healthy controls, and all seven meningiomas analyzed displayed loss of the wild-type allele according to the classic two-hit model for tumor-suppressor genes. In silico modeling predicted the variant to affect the tertiary structure of the protein, and functional analyses showed that the activity of the altered SUFU was significantly reduced and therefore led to dysregulated hedgehog (Hh) signaling. SUFU is a known tumor-suppressor gene previously associated with childhood medulloblastoma predisposition. Our genetic and functional analyses indicate that germline mutations in SUFU also predispose to meningiomas, particularly to multiple meningiomas. It is possible that other genic mutations resulting in aberrant activation of the Hh pathway might underlie meningioma predisposition in families with an unknown etiology.
ARID1A has been identified as a novel tumor suppressor gene in ovarian cancer and subsequently in various other tumor types. ARID1A belongs to the ARID domain containing gene family, which comprises ...of 15 genes involved, for example, in transcriptional regulation, proliferation and chromatin remodeling. In this study, we used exome sequencing data to analyze the mutation frequency of all the ARID domain containing genes in 25 microsatellite unstable (MSI) colorectal cancers (CRCs) as a first systematic effort to characterize the mutation pattern of the whole ARID gene family. Genes which fulfilled the selection criteria in this discovery set (mutations in at least 4/25 16% samples, including at least one nonsense or splice site mutation) were chosen for further analysis in an independent validation set of 21 MSI CRCs. We found that in addition to ARID1A, which was mutated in 39% of the tumors (18/46), also ARID1B (13%, 6/46), ARID2 (13%, 6/46) and ARID4A (20%, 9/46) were frequently mutated. In all these genes, the mutations were distributed along the entire length of the gene, thus distinguishing them from typical MSI target genes previously described. Our results indicate that in addition to ARID1A, other members of the ARID gene family may play a role in MSI CRC.
What's new?
ARID1A was recently identified as a novel tumor suppressor gene. In this study, the authors used exome sequencing to analyze mutation frequency in ARID1A and other members of the ARID gene family in microsatellite‐unstable (MSI) colorectal cancer (CRC). They found inactivating mutations in ARID1A in 39% of these tumors. Three other ARID genes (ARID1B, ARID2 and ARID4A) were frequently mutated as well. These results indicate that members of the ARID gene family may play an important role in MSI CRC and other human cancers.
Abstract
Uterine leiomyosarcoma (ULMS) is a highly aggressive smooth muscle tumor with poorly understood pathogenesis. Although ULMS is believed to develop as a de novo tumor, it has been suggested ...that in some cases uterine leiomyoma (UL) could serve as a precursor lesion for the pathogenesis. ULs are extremely common tumors affecting up to 70% of women. Whereas 90% of ULs are classified as conventional ULs, 10% of the tumors represent subtype histopathologies, some of which have features associated with malignancy. These subtypes include tumors with bizarre nuclei and increased cellularity. In this study, we investigated the genetic and molecular background of one ULMS showing simultaneously mixed histopathologies of UL with bizarre nuclei and cellular UL.
The study material consisted of four formalin-fixed paraffin-embedded samples having distinct areas of ULMS, UL with bizarre nuclei, and cellular UL. Fallopian tube sample was used as a normal control. Libraries for exome sequencing were prepared with KAPA Library Preparation Kit and subjected to exome capture with NimbleGen SeqCap EZ System (Roche). Paired-end sequencing was performed with HiSeq2500 (Illumina). SNP genotyping data was produced with Infinium Omni2.5-8 Kit (Illumina). Immunohistochemistry was performed to proteins commonly displaying aberrant expression in UL and/or ULMS. Telomere-specific fluorescence in situ hybridization was conducted to detect alternatively lengthened telomeres (ALT), a feature often associated with ULMS pathogenesis.
Exome sequencing data and protein expression analyses revealed that all tumor compartments were negative for UL driver alterations, including MED12 mutations, HMGA2 overexpression and biallelic FH inactivation. Data from exome sequencing indicated that ULMS shared the largest number of variants with UL with bizarre nuclei, including the pathogenic splice site mutation c.994-1G>A in tumor protein 53 (TP53). SNP genotyping data showed identical and large chromosomal aberrations in 6q, 9p, 10q and 19q in ULMS and UL with bizarre nuclei. No such aberrations were shared between ULMS and cellular UL. All tumor compartments shared changes in chromosomes 1q, 2p, 12q, 13q, 17q and 17p. ULMS showed prominent ALT, whereas UL with bizarre nuclei had a weaker ALT phenotype. Cellular UL had normal telomere lengths.
In conclusion, these results suggest that the genome, including telomeres, of UL with bizarre nuclei resembles that of ULMS. Large genomic events have probably occurred in UL with bizarre nuclei during tumor evolution, leading to unstable chromosomes, which in turn favored the development of ULMS. Also, all tumor compartments displayed shared genetic variation, suggesting a common evolutionary origin. Understanding the pathogenesis of ULMS and the malignant potential of ULs is clinically highly relevant, as it may improve the diagnosis, prevent malignant transformation and enable the design of new treatments.
Citation Format: Netta Mäkinen, Terhi Ahvenainen, Pernilla von Nandelstadh, Ralf Bützow, Pia Vahteristo. Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5383.
Abstract
Uterine adenomyosis is a condition in which ectopic endometrial glands are present in myometrial stroma surrounded by smooth muscle cell hyperplasia. Foci of adenomyosis growing as a tumor ...like mass are called adenomyomas. Uterine adenomyomas are common tumors and they share symptoms, including pelvic pain and abnormal bleeding, with uterine leiomyomas. The two tumor types are challenging to distinguish from one another and the diagnosis is usually confirmed only after surgery by pathological evaluation. The molecular background of uterine adenomyomas is not currently well known. In uterine leiomyomas, somatic mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) protein overexpression, and fumarate hydratase (FH) inactivation are well established as major mutually exclusive driver events covering 80-90% of the tumors. Here, we have analyzed the presence of these changes in a set of 21 uterine adenomyomas. Representative areas of formalin-fixed paraffin embedded archival uterine adenomyoma tissue samples were used to construct a tissue microarray. The HMGA2 overexpression and the FH inactivation were assessed using immunohistochemistry with anti HMGA and 2SC antibodies, respectively. DNA was extracted from the tumor samples to determine the MED12 mutation status by direct sequencing of exons 1 and 2 of the gene. MED12 c.131GA, p.G44D mutation was found in two adenomyoma samples out of 21 (9.5%). Strong positive staining of 2SC indicating FH inactivation was present in one sample which also showed reduced FH protein expression when validated with an independent method using anti-FH immunostaining. Sequencing revealed a frameshift mutation c.911delC, p.P304fs in exon 7 leading to a premature stop codon 25 codons later. The mutation was also found in a separate uterine leiomyoma of the same patient and both tumor samples mostly presented the mutant allele indicating loss of heterozygosity of the wild type allele. This, together with the patient's medical history of previous uterine leiomyomas, indicates the germline origin of the mutation and thus a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. No changes in HMGA2 expression were detected with all samples presenting normal expression levels. In conclusion, MED12 mutations are present in a subset of uterine adenomyomas. The mutation frequency of 9.5% that was observed here in our adenomyoma sample series is considerably lower than that of 70% in uterine leiomyomas. Our results also suggest that adenomyomas may be linked to HLRCC in which they have not been previously reported. The driver events behind uterine adenomyomas remain mostly unknown and further large-scale studies are warranted to clarify the spectrum of underlying mutations and molecular background of these common tumors.
Citation Format: Tuomas A. Heikkinen, Anna Äyräväinen, Janne Hänninen, Terhi Ahvenainen, Ralf Bützow, Annukka Pasanen, Pia Vahteristo. Uterine leiomyoma driver events in uterine adenomyomas abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4611.
Gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer mortality. Three hereditary gastric cancer syndromes have been described; hereditary diffuse gastric ...cancer (HDGC), familial intestinal gastric cancer (FIGC) and gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS). Thirty per cent of HDGC families have heterozygous germline mutations in
CDH1
, which encodes E-cadherin. A germline truncating mutation in the gene encoding α-E-catenin (
CTNNA1
) was also recently discovered in a family with HDGC, but no other genes specifically predisposing to gastric cancer have been identified, leaving the majority of cases showing familial aggregation without a known genetic cause. The aim of this study was to find the putative gastric cancer predisposing gene defect in a family with HDGC that had previously been tested negative for mutations in
CDH1
. In this family, there were six cases of diffuse gastric cancer in two generations. Exome sequencing was applied to two affected family members. The shared variants which were predicted deleterious in silico and could not be found in databases or in a control set of over 4,000 individuals were Sanger sequenced in a third family member. Three candidate variants were identified: p.Glu1313Lys in
Insulin receptor
(
INSR
), p.Arg81Pro in
F
-
box protein 24
(
FBXO24
) and p.Pro1146Leu in
DOT1
-
like histone H3K79 methyltransferase
(
DOT1L
). These variants and adjacent regions were screened for in an additional 26 gastric cancer patients with a confirmed (n = 13) or suspected (n = 13) family history of disease, but no other non-synonymous mutations were identified. This study identifies
INSR
,
FBXO24
and
DOT1L
as new candidate diffuse gastric cancer susceptibility genes, which should be validated in other populations. Of these genes,
INSR
is of special interest as insulin signaling was recently shown to affect tumor cell invasion capability by modulating E-cadherin glycosylation.
PDF
