Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

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 infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. 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 complex4, 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 mice5. 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.

Molecular subclass of uterine fibroids predicts tumor shrinkage in response to ulipristal acetate.

Precision medicine carries great potential for management of all tumor types. The aim of this retrospective study was to investigate if the two most common genetically distinct uterine fibroid subclasses, driven by aberrations in MED12 and HMGA2 genes, respectively, influence response to treatment with the progesterone receptor modulator ulipristal acetate. Changes in diameter and mutation status were derived for 101 uterine fibroids surgically removed after ulipristal acetate treatment. A significant difference in treatment response between the two major subclasses was detected. MED12 mutant fibroids had 4.4 times higher odds of shrinking in response to ulipristal acetate treatment as compared to HMGA2 driven fibroids (95% confidence interval 1.37-13.9; P = 0.013), and in a multivariate analysis molecular subclassification was an independent predictive factor. Compatible with this finding, gene expression and DNA methylation analyses revealed subclass specific differences in progesterone receptor signaling. The work provides a proof-of-principle that uterine fibroid treatment response is influenced by molecular subclass and that the genetic subclasses should be taken into account when evaluating current and future uterine fibroid therapies.

Exome-wide somatic mutation characterization of small bowel adenocarcinoma.

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (AI) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.

Patterns of chromosome 18 loss of heterozygosity in multifocal ileal neuroendocrine tumors.

Ileal neuroendocrine tumors (NETs) represent the most common neoplasm of the small intestine. Although up to 50% of patients with ileal NETs are diagnosed with multifocal disease, the mechanisms by which multifocal ileal NETs arise are not yet understood. In this study, we analyzed genome-wide sequencing data to examine patterns of copy number variation in 40 synchronous primary ileal NETs derived from three patients. Chromosome (chr) 18 loss of heterozygosity (LOH) was the most frequent copy number alteration identified; however, not all primary tumors from the same patient had evidence of this LOH. Our data revealed three distinct patterns of chr18 allelic loss, indicating that primary tumors from the same patient can present different LOH patterns including retention of either parental allele. In conclusion, our results are consistent with the model that multifocal ileal NETs originate independently. In addition, they suggest that there is no specific germline allele on chr18 that is the target of somatic LOH.

Exome and immune cell score analyses reveal great variation within synchronous primary colorectal cancers.

BACKGROUND: Approximately 4% of colorectal cancer (CRC) patients have at least two simultaneous cancers in the colon. Due to the shared environment, these synchronous CRCs (SCRCs) provide a unique setting to study colorectal carcinogenesis. Understanding whether these tumours are genetically similar or distinct is essential when designing therapeutic approaches. METHODS: We performed exome sequencing of 47 primary cancers and corresponding normal samples from 23 patients. Additionally, we carried out a comprehensive mutational signature analysis to assess whether tumours had undergone similar mutational processes and the first immune cell score analysis (IS) of SCRC to analyse the interplay between immune cell invasion and mutation profile in both lesions of an individual. RESULTS: The tumour pairs shared only few mutations, favouring different mutations in known CRC genes and signalling pathways and displayed variation in their signature content. Two tumour pairs had discordant mismatch repair statuses. In majority of the pairs, IS varied between primaries. Differences were not explained by any clinicopathological variable or mutation burden. CONCLUSIONS: The study shows major diversity within SCRCs. Rather than rely on data from one tumour, our study highlights the need to evaluate both tumours of a synchronous pair for optimised targeted therapy.

Exome Sequencing of Uterine Leiomyosarcomas Identifies Frequent Mutations in TP53, ATRX, and MED12.

Uterine leiomyosarcomas (ULMSs) are aggressive smooth muscle tumors associated with poor clinical outcome. Despite previous cytogenetic and molecular studies, their molecular background has remained elusive. To examine somatic variation in ULMS, we performed exome sequencing on 19 tumors. Altogether, 43 genes were mutated in at least two ULMSs. Most frequently mutated genes included tumor protein P53 (TP53; 6/19; 33%), alpha thalassemia/mental retardation syndrome X-linked (ATRX; 5/19; 26%), and mediator complex subunit 12 (MED12; 4/19; 21%). Unlike ATRX mutations, both TP53 and MED12 alterations have repeatedly been associated with ULMSs. All the observed ATRX alterations were either nonsense or frameshift mutations. ATRX protein levels were reliably analyzed by immunohistochemistry in altogether 44 ULMSs, and the majority of tumors (23/44; 52%) showed clearly reduced expression. Loss of ATRX expression has been associated with alternative lengthening of telomeres (ALT), and thus the telomere length was analyzed with telomere-specific fluorescence in situ hybridization. The ALT phenotype was confirmed in all ULMSs showing diminished ATRX expression. Exome data also revealed one nonsense mutation in death-domain associated protein (DAXX), another gene previously associated with ALT, and the tumor showed ALT positivity. In conclusion, exome sequencing revealed that TP53, ATRX, and MED12 are frequently mutated in ULMSs. ALT phenotype was commonly seen in tumors, indicating that ATR inhibitors, which were recently suggested as possible new drugs for ATRX-deficient tumors, could provide a potential novel therapeutic option for ULMS.

Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers.

Uterine leiomyomas are common benign smooth muscle tumors that impose a major burden on women's health. Recent sequencing studies have revealed recurrent and mutually exclusive mutations in leiomyomas, suggesting the involvement of molecularly distinct pathways. In this study, we explored transcriptional differences among leiomyomas harboring different genetic drivers, including high mobility group AT-hook 2 (HMGA2) rearrangements, mediator complex subunit 12 (MED12) mutations, biallelic inactivation of fumarate hydratase (FH), and collagen, type IV, alpha 5 and collagen, type IV, alpha 6 (COL4A5-COL4A6) deletions. We also explored the transcriptional consequences of 7q22, 22q, and 1p deletions, aiming to identify possible target genes. We investigated 94 leiomyomas and 60 corresponding myometrial tissues using exon arrays, whole genome sequencing, and SNP arrays. This integrative approach revealed subtype-specific expression changes in key driver pathways, including Wnt/ß-catenin, Prolactin, and insulin-like growth factor (IGF)1 signaling. Leiomyomas with HMGA2 aberrations displayed highly significant up-regulation of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1), suggesting that HMGA2 promotes tumorigenesis through PLAG1 activation. This was supported by the identification of genetic PLAG1 alterations resulting in expression signatures as seen in leiomyomas with HMGA2 aberrations. RAD51 paralog B (RAD51B), the preferential translocation partner of HMGA2, was up-regulated in MED12 mutant lesions, suggesting a role for this gene in the genesis of leiomyomas. FH-deficient leiomyomas were uniquely characterized by activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes, supporting the hypothesis that accumulation of fumarate leads to activation of the oncogenic transcription factor NRF2. This study emphasizes the need for molecular stratification in leiomyoma research and possibly in clinical practice as well. Further research is needed to determine whether the candidate biomarkers presented herein can provide guidance for managing the millions of patients affected by these lesions.

Loss of ATRX/DAXX expression and alternative lengthening of telomeres in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas (ULs) are the most common gynecologic tumors and affect 3 of every 4 women by the age of 50 years. The majority of ULs are classified as conventional tumors, whereas 10% represent various histopathological subtypes with features that mimic malignancy. These subtypes include cellular and mitotically active ULs and ULs with bizarre nuclei. Uterine leiomyosarcoma (ULMS), the malignant counterpart of UL, is an aggressive cancer with poor overall survival. The early diagnosis and preoperative differentiation of ULMS from UL are often challenging because their symptoms and morphology resemble one another. Recent studies have shown frequent loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein (DAXX) expression in ULMS, and this is often associated with an alternative lengthening of telomeres (ALT) phenotype. METHODS: To investigate ATRX and DAXX expression and the presence of ALT in UL subtypes, immunohistochemical and telomere-specific fluorescence in situ hybridization analyses were performed. The study material consisted of 142 formalin-fixed, paraffin-embedded tissue samples representing various UL subtypes and 64 conventional ULs. RESULTS: A loss of ATRX or DAXX and/or ALT was detected in 6.3% of the histopathological UL subtype samples (9 of 142). Two patients whose ULs showed either ATRX loss or ALT were later diagnosed with a pulmonary smooth muscle tumor. Pulmonary tumors displayed molecular alterations found in the corresponding uterine tumors, which indicated metastasis to the lungs. All conventional ULs displayed normal ATRX, DAXX, and telomeres. CONCLUSIONS: These results highlight the differences between conventional and histopathologically atypical ULs and indicate that some UL subtype tumors may harbor long-term malignant potential.

Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry.

MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5' end nonsense mutation (c.97G>T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin-α and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD.

Characterization of MED12, HMGA2, and FH alterations reveals molecular variability in uterine smooth muscle tumors.

Uterine smooth muscle tumors range from benign leiomyomas to malignant leiomyosarcomas. Based on numerous molecular studies, leiomyomas and leiomyosarcomas mostly lack shared mutations and the majority of tumors are believed to develop through distinct mechanisms. To further characterize the molecular variability among uterine smooth muscle tumors, and simultaneously insinuate their potential malignant progression, we examined the frequency of known genetic leiomyoma driver alterations (MED12 mutations, HMGA2 overexpression, biallelic FH inactivation) in 65 conventional leiomyomas, 94 histopathological leiomyoma variants (18 leiomyomas with bizarre nuclei, 22 cellular, 29 highly cellular, and 25 mitotically active leiomyomas), and 51 leiomyosarcomas. Of the 210 tumors analyzed, 107 had mutations in one of the three driver genes. No tumor had more than one mutation confirming that all alterations are mutually exclusive. MED12 mutations were the most common alterations in conventional and mitotically active leiomyomas and leiomyosarcomas, while leiomyomas with bizarre nuclei were most often FH deficient and cellular tumors showed frequent HMGA2 overexpression. Highly cellular leiomyomas displayed the least amount of alterations leaving the majority of tumors with no known driver aberration. Our results indicate that based on the molecular background, histopathological leiomyoma subtypes do not only differ from conventional leiomyomas, but also from each other. The presence of leiomyoma driver alterations in nearly one third of leiomyosarcomas suggests that some tumors arise through leiomyoma precursor lesion or that these mutations provide growth advantage also to highly aggressive cancers. It is clinically relevant to understand the molecular background of various smooth muscle tumor subtypes, as it may lead to improved diagnosis and personalized treatments in the future.

Clonally related uterine leiomyomas are common and display branched tumor evolution.

Uterine leiomyomas are extremely frequent benign smooth muscle tumors often presenting as multiple concurrent lesions and causing symptoms such as abnormal menstrual bleeding, abdominal pain and infertility. While most leiomyomas are believed to arise independently, a few studies have encountered separate lesions harboring identical genetic changes, suggesting a common clonal origin. To investigate the frequency of clonally related leiomyomas, genome-wide tools need to be utilized, and thus little is known about this phenomenon. Using MED12 sequencing and SNP arrays, we searched for clonally related uterine leiomyomas in a set of 103 tumors from 14 consecutive patients who entered hysterectomy owing to symptomatic lesions. Whole-genome sequencing was also utilized to study the genomic architecture of clonally related tumors. This revealed four patients to have two or more tumors that were clonally related, all of which lacked MED12 mutations. Furthermore, some tumors were composed of genetically distinct subclones, indicating a nonlinear, branched model of tumor evolution. DEPDC5 was discovered as a novel tumor suppressor gene playing a role in the progression of uterine leiomyomas. Perhaps counterintuitively­considering Knudson's two-hit hypothesis­a large shared deletion was followed by different truncating DEPDC5 mutations in four clonally related leiomyomas. This study provides insight into the intratumor heterogeneity of these tumors and suggests that a shared clonal origin is a common feature of leiomyomas that do not carry an MED12 mutation. These observations also offer one explanation to the common occurrence of multiple concurrent lesions.

Global metabolomic profiling of uterine leiomyomas.

BACKGROUND: Uterine leiomyomas can be classified into molecularly distinct subtypes according to their genetic triggers: MED12 mutations, HMGA2 upregulation, or inactivation of FH. The aim of this study was to identify metabolites and metabolic pathways that are dysregulated in different subtypes of leiomyomas. METHODS: We performed global metabolomic profiling of 25 uterine leiomyomas and 17 corresponding myometrium specimens using liquid chromatography-tandem mass spectroscopy. RESULTS: A total of 641 metabolites were detected. All leiomyomas displayed reduced homocarnosine and haeme metabolite levels. We identified a clearly distinct metabolomic profile for leiomyomas of the FH subtype, characterised by metabolic alterations in the tricarboxylic acid cycle and pentose phosphate pathways, and increased levels of multiple lipids and amino acids. Several metabolites were uniquely elevated in leiomyomas of the FH subtype, including N6-succinyladenosine and argininosuccinate, serving as potential biomarkers for FH deficiency. In contrast, leiomyomas of the MED12 subtype displayed reduced levels of vitamin A, multiple membrane lipids and amino acids, and dysregulation of vitamin C metabolism, a finding which was also compatible with gene expression data. CONCLUSIONS: The study reveals the metabolomic heterogeneity of leiomyomas and provides the requisite framework for strategies designed to target metabolic alterations promoting the growth of these prevalent tumours.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms.

BACKGROUND: Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. METHODS: To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. RESULTS: In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. CONCLUSIONS: Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis. METHODS: MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA). RESULTS: Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours. CONCLUSIONS: Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Characterization of uterine leiomyomas by whole-genome sequencing.

BACKGROUND: Uterine leiomyomas are benign but affect the health of millions of women. A better understanding of the molecular mechanisms involved may provide clues to the prevention and treatment of these lesions. METHODS: We performed whole-genome sequencing and gene-expression profiling of 38 uterine leiomyomas and the corresponding myometrium from 30 women. RESULTS: Identical variants observed in some separate tumor nodules suggested that these nodules have a common origin. Complex chromosomal rearrangements resembling chromothripsis were a common feature of leiomyomas. These rearrangements are best explained by a single event of multiple chromosomal breaks and random reassembly. The rearrangements created tissue-specific changes consistent with a role in the initiation of leiomyoma, such as translocations of the HMGA2 and RAD51B loci and aberrations at the COL4A5-COL4A6 locus, and occurred in the presence of normal TP53 alleles. In some cases, separate events had occurred more than once in single tumor-cell lineages. CONCLUSIONS: Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).

Exomic landscape of MED12 mutation-negative and -positive uterine leiomyomas.

Uterine leiomyomas are extremely common tumors originating from the smooth muscle cells of myometrium. We recently reported recurrent somatic mutations in mediator complex subunit 12 (MED12) in the majority of these lesions, and analyzed chromosomal abnormalities in leiomyomas by whole-genome sequencing. The aim of our study was to examine in detail uterine leiomyoma exomes, to search for driver mutations in MED12 mutation-negative leiomyomas and to scrutinize MED12 mutation-positive leimyomas for additional contributing mutations. We analyzed whole exome sequencing data of 27 uterine leiomyomas (12 MED12 mutation-negative and 15 MED12 mutation-positive) and their paired normal myometrium. We searched for genes, which would be recurrently mutated. No such genes were identified in MED12 mutation-negative uterine leiomyomas. Similarly, MED12 mutation-positive leiomyomas displayed no additional recurrent changes. The complete lack of novel driver point mutations in the examined series highlights the unique role of MED12 mutations in genesis of uterine leiomyomas, and suggests that these mutations alone may be sufficient for tumor development. Additional factors that cannot be detected by exome sequencing, such as somatic structural rearrangements, epigenetic events and intronic variants, are likely to have a particular impact to the development of MED12 wild-type lesions.

Genomic insights into the mechanisms of FGFR1 dependency in squamous cell lung cancer.

Although subsets of patients with lung squamous cell carcinoma (LSCC) benefit from immunotherapy, there are few effective molecularly targeted treatments for LSCC. Fibroblast growth factor receptor (FGFR) inhibitors provide a therapeutic option for patients with LSCC harboring FGFR aberrations, but their therapeutic efficacy has been limited to date. In this issue of the JCI, Malchers et al. identified tail-to-tail rearrangements, either within or near FGFR1, that are associated with FGFR1 dependency and sensitivity to FGFR inhibition in LSCC. These results may help improve the selection of patients with LSCC who are most likely to benefit from treatment with FGFR inhibitors.

Comparison of 2SC, AKR1B10, and FH Antibodies as Potential Biomarkers for FH-deficient Uterine Leiomyomas.

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.

Whole genome sequencing reveals the independent clonal origin of multifocal ileal neuroendocrine tumors.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) are the most common neoplasms of the small bowel. The majority of tumors are located in the distal ileum with a high incidence of multiple synchronous primary tumors. Even though up to 50% of SI-NET patients are diagnosed with multifocal disease, the mechanisms underlying multiple synchronous lesions remain elusive. METHODS: We performed whole genome sequencing of 75 de-identified synchronous primary tumors, 15 metastases, and corresponding normal samples from 13 patients with multifocal ileal NETs to identify recurrent somatic genomic alterations, frequently affected signaling pathways, and shared mutation signatures among multifocal SI-NETs. Additionally, we carried out chromosome mapping of the most recurrent copy-number alterations identified to determine which parental allele had been affected in each tumor and assessed the clonal relationships of the tumors within each patient. RESULTS: Absence of shared somatic variation between the synchronous primary tumors within each patient was observed, indicating that these tumors develop independently. Although recurrent copy-number alterations were identified, additional chromosome mapping revealed that tumors from the same patient can gain or lose different parental alleles. In addition to the previously reported CDKN1B loss-of-function mutations, we observed potential loss-of-function gene alterations in TNRC6B, a candidate tumor suppressor gene in a small subset of ileal NETs. Furthermore, we show that multiple metastases in the same patient can originate from either one or several primary tumors. CONCLUSIONS: Our study demonstrates major genomic diversity among multifocal ileal NETs, highlighting the need to identify and remove all primary tumors, which have the potential to metastasize, and the need for optimized targeted treatments.

Genomic Evolution in a Patient With Lung Adenocarcinoma With a Germline EGFR T790M Mutation.

INTRODUCTION: A subset of lung adenocarcinomas (ADs) has been found to have somatic activating mutations in the tyrosine kinase domain of the EGFR gene, associated with response to EGFR tyrosine kinase inhibitor therapy. Rare germline mutations within this domain, including EGFR T790M, have been associated with genetic susceptibility to lung ADs. Using high-throughput sequencing, we elucidate the genomic evolution in tissues from a patient with lung AD carrying a germline EGFR T790M mutation. METHODS: We performed microdissection, targeted panel, and whole-exome sequencing to molecularly characterize multiple foci of atypical adenomatous hyperplasia (AAH), in situ and invasive components of AD, normal lung tissue, and whole blood from the patient. Normal lung tissue was analyzed for potential acquired somatic genome alterations ("field effect"). RESULTS: All lesions harbored a secondary somatic EGFR mutation, either L858R or L861Q, in addition to the germline T790M mutation. Clear overlap was observed between the somatic profiles of in situ and invasive AD components, confirming clonal relatedness. AAH lesions shared few to no somatic alterations with the AD, suggesting clonal independence. No robust evidence of field effect was identified in the normal lung tissue. CONCLUSIONS: Somatic EGFR mutations are early events in the pathogenesis of lung ADs arising in the context of germline EGFR T790M. Synchronous AAH lesions seem to be independent. Stepwise genomic evolution is observed in association with invasiveness of the neoplastic cell population.