Lung cancer has a high mortality rate in men and women worldwide. Approximately 15% of diagnosed patients with this type of cancer do not exceed the 5-year survival rate. Unfortunately, diagnosis is established in advanced stages, where other tissues or organs can be affected. In recent years, lineage-specific transcription factors have been associated with a variety of cancers. One such transcription factor possibly regulating cancer is RUNX2, the master gene of early and late osteogenesis. In thyroid and prostate cancer, it has been reported that RUNX2 regulates expression of genes important in tumor cell migration and invasion. In this study, we report on RUNX2/ p57 overexpression in 16 patients with primary non-small cell lung cancer and/or metastatic lung cancer associated with H3K27Ac at P1 gene promoter region. In some patients, H3K4Me3 enrichment was also detected, in addition to WDR5, MLL2, MLL4, and UTX enzyme recruitment, members of the COMPASS-LIKE complex. Moreover, transforming growth factor-β induced RUNX2/ p57 overexpression and specific RUNX2 knockdown supported a role for RUNX2 in epithelial mesenchymal transition, which was demonstrated through loss of function assays in adenocarcinoma A549 lung cancer cell line. Furthermore, RUNX2 increased expression of epithelial mesenchymal transition genes VIMENTIN, TWIST1, and SNAIL1, which reflected increased migratory capacity in lung adenocarcinoma cells.

Parathyroid carcinoma (PC) is a rare endocrine malignancy, accounting for <1% of all cases of sporadic primary hyperparathyroidism (PHPT) and up to 15% in the hereditary hyperparathyroidism-jaw tumor syndrome. Genomic alterations identified in PC are mostly represented by CDC73 gene mutations, codifying for a loss-of-function protein termed parafibromin. Whole exome sequencing identified mutations in other genes, such as mTOR, KMT2D, CDKN2C, THRAP3, PIK3CA, and EZH2 genes, CCND1 gene amplification. The diagnosis of PC is quite difficult due to the lack of reliable clinical diagnostic criteria, and in the majority of cases is made postoperatively at histological examination. The clinical manifestations of PC are primarily due to the excessive secretion of PTH by the tumor rather than spread to local or distant organs. En bloc resection of the parathyroid tumor represents the initial mainstay treatment of patients with PC. Multiple surgical procedures may be required, although surgical morbidity should be taken into account. A 5- and 10-year survival between 77-100 and 49-91%, respectively, has been reported. When the tumor is no more resectable, medical treatment of hypercalcemia has a pivotal role in the management of these patients.

Human papillomavirus (HPV) is a necessary but insufficient cause of a subset of oral squamous cell carcinomas (OSCCs) that is increasing markedly in frequency. To identify contributory, secondary genetic alterations in these cancers, we used comprehensive genomics methods to compare 149 HPV-positive and 335 HPV-negative OSCC tumor/normal pairs. Different behavioral risk factors underlying the two OSCC types were reflected in distinctive genomic mutational signatures. In HPV-positive OSCCs, the signatures of APOBEC cytosine deaminase editing, associated with anti-viral immunity, were strongly linked to overall mutational burden. In contrast, in HPV-negative OSCCs, T>C substitutions in the sequence context 5'-ATN-3' correlated with tobacco exposure. Universal expression of HPV

Natural Killer (NK) cells suppress tumor initiation and metastasis. Most carcinomas are heterogeneous mixtures of epithelial, mesenchymal and hybrid tumor cells, but the relationships of these phenotypes to NK susceptibility are understood incompletely. Grainyhead-like-2 (GRHL2) is a master programmer of the epithelial phenotype, that is obligatorily down-regulated during experimentally induced Epithelial-Mesenchymal Transition (EMT). Here, we utilize GRHL2 re-expression to discover unifying molecular mechanisms that link the epithelial phenotype with NK-sensitivity. GRHL2 enhanced the expression of ICAM-1, augmenting NK-target cell synaptogenesis and NK killing of target cells. The expression of multiple interferon response genes, including ICAM1, anti-correlated with EMT. We identified two novel GRHL2-interacting proteins, the histone methyltransferases KMT2C and KMT2D. Mesenchymal-epithelial transition, NK-sensitization and ICAM-1 expression were promoted by GRHL2-KMT2C/D interactions and by GRHL2 inhibition of p300, revealing novel and potentially targetable epigenetic mechanisms connecting the epithelial phenotype with target cell susceptibility to NK killing.

PURPOSE: We aimed to investigate the genomic profile of breast sarcomas (BS) and compare with that of malignant phyllodes tumours (MPT).
METHODS: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens from 17 cases of BS diagnosed at Singapore General Hospital from January 1991 to December 2014. Targeted deep sequencing and copy number variation (CNV) analysis on 16 genes, which included recurrently mutated genes in phyllodes tumours and genes associated with breast cancer, were performed on these samples. Genetic alterations (GA) observed were summarised and analysed.
RESULTS: Nine cases met the quality control requirements for both targeted deep sequencing and CNV analysis. Three (33.33%) were angiosarcomas and 6 (66.67%) were non-angiosarcomas. In the non-angiosarcoma group, 83.33% (n = 5) of the patients had GA in the TERT gene. The other commonly mutated genes in this group of tumours were MED12 (n = 4, 66.67%), BCOR (n = 4, 66.67%), KMT2D (n = 3, 50%), FLNA (n = 3, 50%) and NF1 (n = 3, 50%). In contrast, none of the angiosarcomas had mutations or copy number alterations in TERT, MED12, BCOR, FLNA or NF1. Eighty percent of patients with GA in TERT (n = 5) had concurrent mutations in MED12. Sixty percent (n = 3) of these cases also demonstrated GA in NF1, PIK3CA or EGFR which are known cancer driver genes.
CONCLUSIONS: The non-angiosarcoma group of BS was found to share similar GA as those described for MPT, which may suggest a common origin and support their consideration as a similar group of tumours with regard to management and prognostication.

RATIONALE: Follicular dendritic cell (FDC) sarcoma is a rare tumor with FDC differentiation that typically arises within lymph nodes but can also occur extranodally. To date, the primary esophageal FDC sarcoma has not been reported in the English literature.
PATIENT CONCERNS: We described a 67-year-old female who foremostly presented with dysphagia, and the patient was readmitted due to a dry cough and pain of his right shoulder 2 years after initial treatment.
DIAGNOSES: Primary esophageal FDC sarcoma with the right superior mediastinal lymph node metastasis.
INTERVENTIONS: The esophageal tumor was removed by endoscopic submucosal dissection at the first hospitalization. At the second hospitalization 2 years after the initial visit, the tracheal stent loaded with (125) iodine radioactive seeds was placed. The profiles of genetic variations and immunotherapeutic biomarkers were also explored by next-generation sequencing protocol from the patient's blood, esophageal primary, and mediastinal metastatic tumor samples.
OUTCOMES: The patient's symptom transitorily relieved, but she gave up further treatment and died 2 months after the tracheal stent was placed. As for the genomic alterations, we found 9 gene mutations in all the samples, including checkpoint kinase 2(CHEK2), FAT atypical cadherin 1 (FAT1), tumor protein 53 (TP53), DPYD, ERBB2 interacting protein (ERBB2IP), FBXW7, KMT2D, PPP2R1A, TSC2, whereas amplification of MYC was only in the metastatic example. The analysis of clonal evolution and phylogenetic tree showed the propagation and replay of polyclonal esophageal FDC sarcoma. At the same time, the detection of biomarkers for immunotherapy revealed microsatellite stable and mismatch repair-proficient (pMMR), which predicted a relatively poor anti-programmed death (PD-1)/programmed death ligand (PD-L1) immunotherapy outcome. On the contrary, the tumor mutational burdens were 10 mutations per 1 million bases in both the primary and metastatic tumor sample, which ranked the top 23.3% in solid tumors mutational burdens database of Geneseeq and might be a good predictor of the efficacy of anti-PD-1/PD-L1 immunotherapy.
LESSONS: To the best of our knowledge, this case report announced the first case of extranodal primary esophageal FDC sarcoma in the world, and firstly revealed its unique genetic alterations profiles, which might contribute to further in-depth study of this rare disease.

Determining the clinical significance of germline and somatic KMT2D missense variants (MVs) in Kabuki syndrome (KS) and cancers can be challenging. We analysed 1920 distinct KMT2D MVs that included 1535 germline MVs in controls (Control-MVs), 584 somatic MVs in cancers (Cancer-MVs) and 201 MV in individuals with KS (KS-MVs). The proportion of MVs likely to affect splicing was significantly higher for Cancer-MVs and KS-MVs than in Control-MVs (p = 0.000018). Our analysis identified significant clustering of Cancer-MVs and KS-MVs in the PHD#3 and #4, RING#4 and SET domains. Areas of enrichment restricted to just Cancer-MVs (FYR-C and between amino acids 3043-3248) or KS-MVs (coiled-coil#5, FYR-N and between amino acids 4995-5090) were also found. Cancer-MVs and KS-MVs tended to affect more conserved residues (lower BLOSUM scores, p < 0.001 and p = 0.007). KS-MVs are more likely to increase the energy for protein folding (higher ELASPIC ∆∆G scores, p = 0.03). Cancer-MVs are more likely to disrupt protein interactions (higher StructMAn scores, p = 0.019). We reclassify several presumed pathogenic MVs as benign or as variants of uncertain significance. We raise the possibility of as yet unrecognised 'non-KS' phenotype(s) associated with some germline pathogenic KMT2D MVs. Overall, this work provides insights into the disease mechanism of KMT2D variants and can be extended to other genes, mutations in which also cause developmental syndromes and cancer.

The molecular pathogenesis of follicular lymphoma (FL) was partially revealed 3 decades ago, with the discovery of the translocation that brings BCL2 under the influence of immunoglobulin heavy chain enhancers in a vast majority of cases. Despite the importance of this seminal observation, it has become increasingly clear that additional genetic alterations need to occur to trigger neoplastic transformation and disease progression. The evolution of FL involves developmental arrest and disruption of the normal function of one or more of epigenetic regulators including KMT2D/MLL2, EZH2, CBP/CREBBP, p300/EP300, and HIST1H1 in >95% of cases. B-cells "arrested" in germinal centers acquire dozens of additional genetic aberrations that influence key pathways controlling their physiological development including B Cell Receptor (BCR) signaling, PI3K/AKT, TLR, mTOR, NF-κB, JAK/STAT, MAPK, CD40/CD40L, chemokine, and interleukin signaling. Additionally, most cases of FL do not result from linear accumulation of genomic aberrations, but rather evolve from a common progenitor cell population by diverse evolution, creating multiple FL subclones in one patient. Moreover, one of the subclones might acquire a combination of aberrations involving genes controlling cell survival and proliferation including MDM2, CDKN2A/B, BCL6, MYC, TP53, β2M, FOXO1, MYD88, STAT3, or miR-17-92, and this can lead to the transformation of an initially indolent FL to an aggressive lymphoma (2%-3% risk per year). The complexity of the disease is also underscored by the importance of its interactions with the microenvironment that can substantially influence disease development and prognosis. Interpreting individual aberrations in relation to their impact on normal processes, their frequency, position in the disease evolution, and the consequences of their (co)occurrence, are the basis for understanding FL pathogenesis. This is necessary for the identification of patients with risk of early progression or transformation, for the development of novel targeted therapies, and for personalized treatment approaches. In this review, we summarize recent knowledge of molecular pathways and microenvironmental components involved in FL biology, and discuss them in the context of physiological B-cell development, FL evolution, and targeted therapies.

OBJECTIVE: Despite advances in the identification of epigenetic alterations in pancreatic cancer, their biological roles in the pathobiology of this dismal neoplasm remain elusive. Here, we aimed to characterise the functional significance of histone lysine methyltransferases (KMTs) and demethylases (KDMs) in pancreatic tumourigenesis.
DESIGN: DNA methylation sequencing and gene expression microarrays were employed to investigate CpG methylation and expression patterns of KMTs and KDMs in pancreatic cancer tissues versus normal tissues. Gene expression was assessed in five cohorts of patients by reverse transcription quantitative-PCR. Molecular analysis and functional assays were conducted in genetically modified cell lines. Cellular metabolic rates were measured using an XF24-3 Analyzer, while quantitative evaluation of lipids was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Subcutaneous xenograft mouse models were used to evaluate pancreatic tumour growth in vivo.
RESULTS: We define a new antitumorous function of the histone lysine (K)-specific methyltransferase 2D (KMT2D) in pancreatic cancer.
CONCLUSION: Together our findings define a new tumour suppressor function of KMT2D through the regulation of glucose/fatty acid metabolism in pancreatic cancer.

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

Primary central nervous system lymphoma (PCNSL) is a rare and special type of non-Hodgkin lymphoma. The treatment of PCNSL is comprehensive, combining surgery, radiotherapy, and chemotherapy. However, the outcome is poor because of its high invasiveness and rate of recurrence. We analyzed 22 cases of PCNSL using next-generation sequencing (NGS) to detect 64 candidate genes. We used immunohistochemical methods to analyze gene expression in 57 PCNSL samples. NGS showed that recurrent mutations in KMT2D and CD79B, components of the NF-κB pathway, accounted for 65% of total mutations in PCNSL samples. The most frequent mutated gene was PIM1 (77.27%, 17/22), followed by MYD88 (63.64%, 14/22), CD79B (69.09%, 13/22), and KMT2D (50.00%, 11/22). Mutations of the CD79B gene were associated with an inferior progression-free survival (PFS), and GNA13 gene mutations were associated with a shorter PFS and overall survival (OS) in PCNSL patients (P < .05). PIM1 and MYD88 were highly expressed in PCNSL patients and were related to their OS time. MYD88 overexpression might be an independent and poor prognostic predictor of OS time. In summary, we identified highly recurrent genetic lesions in CD79B and KMT2D, components of the NF-κB pathway, in PCNSL and validated the expression of PIM1 and MYD88 related to poor survival, thereby providing novel insights into the pathogenesis and precision medicine of PCNSL.

PURPOSE: Histone lysine methyltransferase 2D (KMT2D/MLL2) is a known cancer-related protein; however, its function in gastric cancer (GC) remains uncharacterized. The present study sought to investigate the expression pattern and the role of KMT2D in GC.
METHODS: The expression of KMT2D were evaluated at mRNA and protein levels, while its clinico-pathological value were further explored. GC cells were transfected with KMT2D knockdown siRNAs or lentiviruses, and then detected by cell counting kit-8, plate clone formation, cell apoptosis, cycle, migration, invasion, and tumorigenesis assays.
RESULTS: Overexpression of KMT2D was observed in GC samples, and was strongly associated with poor survival. Depletion of KMT2D suppressed cell proliferation and induced apoptosis.
CONCLUSION: Our study demonstrated the upregulation of KMT2D in GC tissue, and KMT2D modulates proliferation and apoptosis in GC. Therefore, KMT2D might represent a novel oncogene for prognosis and optimal treatment of GC patients.

BACKGROUND: We present a rare case where distant metastasis of a low grade bladder tumor was observed. We carried out detailed genomic analysis and cell based experiments on patient tumor samples to study tumor evolution, possible cause of disease and provide personalized treatment strategies.
CASE PRESENTATION: A man with a smoking history was diagnosed with a low-grade urothelial carcinoma of the bladder and a concurrent high-grade upper urinary tract tumor. Seven years later he had a lung metastasis. We carried out exome sequencing on all the patient's tumors and peripheral blood (germline) to identify somatic variants. We constructed a phylogenetic tree to capture how the tumors are related and to identify somatic changes important for metastasis. Although distant metastasis of low-grade bladder tumor is rare, the somatic variants in the tumors and the phylogenetic tree showed that the metastasized tumor had a mutational profile most similar to the low grade urothelial carcinoma. The primary and the metastatic tumors shared several important mutations, including in the KMT2D and the RXRA genes. The metastatic tumor also had an activating MTOR mutation, which may be important for tumor metastasis. We developed a mutational signature to understand the biologic processes responsible for tumor development. The mutational signature suggests that the tumor mutations are associated with tobacco carcinogen exposure, which is concordant with the patient's smoking history. We cultured cells from the lung metastasis to examine proliferation and signaling mechanisms in response to treatment. The mTOR inhibitor Everolimus inhibited downstream mTOR signaling and induced cytotoxicity in the metastatic tumor cells.
CONCLUSION: We used genomic analysis to examine a rare case of low grade bladder tumor metastasis to distant organ (lung). Our analysis also revealed exposure to carcinogens found is tobacco as a possible cause in tumor development. We further validated that the patient might benefit from mTOR inhibition as a potential salvage therapy in an adjuvant or recurrent disease setting.

Extranodal natural killer (NK)/T cell lymphoma (ENKTL-NT or NKTCL), with its aggressive nature and poor prognosis, has been widely studied to discover more effective treatment options. Various somatic gene alterations have been identified by traditional Sanger sequencing. However, recently, novel gene mutations in NKTCL have been revealed by next-generation sequencing (NGS) technology, suggesting the potential for novel targeted therapies. This review discusses recurrent aberrations in NKTCL detected by NGS, which can be categorized into three main groups, specifically, tumor suppressors (TP53, DDX3X, and MGA), the JAK/STAT cascade, and epigenetic modifiers (KMT2D, BCOR, ARID1A, and EP300). Some epigenetic dysregulation and DDX3X mutation, which have been rarely identified by traditional sequencing technology, were recently uncovered with high frequencies by NGS. In this review, we summarize the mutational frequencies of various genes in NKTCL. In general, based on our analysis, BCOR is the most frequently mutated gene (16.9%), followed by TP53 (14.7%), and DDX3X (13.6%). The characterization of such genes provides new insight into the pathogenesis of this disease and indicates new biomarkers or therapeutic targets.

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including

OBJECTIVE: We examined expressions of human equilibrative nucleoside transporter-1 (hENT1) and thymidine kinase-1 (TK1), the key enzyme in 4'-[methyl-
METHODS: A total of 19 patients with newly diagnosed gliomas were examined with 4DST PET. Tumor lesions were identified as areas of focally increased uptake, exceeding that of normal brain background. For semi-quantitative analysis, tumor-to-contralateral normal brain tissue (T/N) ratio was determined by dividing the maximal standardized uptake value (SUV) for tumor by that of the mean SUV for reference tissue. The expressions of hENT1, TK1 and Ki-67 in tumor specimens were examined by immunohistochemistry and compared with 4DST T/N ratio.
RESULTS: All but two gliomas showed focally increased 4DST uptake. All gliomas showed hENT1 staining, except one grade II glioma, which was also not visualized on 4DST PET. A significant correlation was observed between T/N ratio and hENT1 score (ρ = 0.90, p < 0.001). All gliomas showed TK1 staining, except two gliomas which were also not visualized on 4DST PET. There was a significant correlation between T/N ratio and TK1 score (ρ = 0.92, p < 0.001). There was a significant correlation between T/N ratio and Ki-67 index (ρ = 0.50, p < 0.03).
CONCLUSION: Results of this preliminary study indicate that expressions of hENT1 and TK1 appear to be important determinants of 4DST uptake in newly diagnosed gliomas.

Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers. Yet, how alterations in H3K4me1 are established and the consequences of these epigenetic changes in tumorigenesis are not well understood. Using ChIP-Seq in human colon cancer cells, we demonstrate that mutant p53 depletion results in decreased H3K4me1 levels at active enhancers that reveal a striking colocalization of mutant p53 and the H3K4 monomethyltransferase MLL4 following chronic tumor necrosis factor alpha (TNFα) signaling. We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNFα-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript (eRNA) synthesis, and mutant p53-dependent target gene activation. Complementary

Adult-type granulosa cell tumors of the ovary (aGCTs) are rare gynecologic malignancies that exhibit a high frequency of somatic FOXL2 c.C402G (p.Cys134Trp) mutation. Treatment of relapsed aGCT remains a significant clinical challenge. Here we show, using whole-exome and cancer gene panel sequencing of 79 aGCTs from two independent cohorts, that truncating mutation of the histone lysine methyltransferase gene KMT2D (also known as MLL2) is a recurrent somatic event in aGCT. Mono-allelic KMT2D-truncating mutations are more frequent in recurrent (10/44, 23%) compared with primary (1/35, 3%) aGCTs (p = 0.02, two-sided Fisher's exact test). IHC detects additional non-KMT2D-mutated aGCTs with loss of nuclear KMT2D expression, suggesting that non-genetic KMT2D inactivation may occur in this tumor type. These findings identify KMT2D inactivation as a novel driver event in aGCTs and suggest that mutation of this gene may increase the risk of disease recurrence.

PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data.
METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry.
RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent.
CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Estrogen receptor alpha (ERα) is a ligand-activated nuclear receptor that directs proliferation and differentiation in selected cancer cell types including mammary-derived carcinomas. These master-regulatory functions of ERα require trans-acting elements such as the pioneer factor FOXA1 to establish a genomic landscape conducive to ERα control. Here, we identify the H3K4 methyltransferase KMT2C as necessary for hormone-driven ERα activity and breast cancer proliferation. KMT2C knockdown suppresses estrogen-dependent gene expression and causes H3K4me1 and H3K27ac loss selectively at ERα enhancers. Correspondingly, KMT2C loss impairs estrogen-driven breast cancer proliferation but has no effect on ER- breast cells. Whereas KMT2C loss disrupts estrogen-driven proliferation, it conversely promotes tumor outgrowth under hormone-depleted conditions. In accordance, KMT2C is one of the most frequently mutated genes in ER-positive breast cancer with KMT2C deletion correlating with significantly shorter progression-free survival on anti-estrogen therapy. From a therapeutic standpoint, KMT2C-depleted cells that develop hormone-independence retain their dependence on ERα, displaying ongoing sensitivity to ERα antagonists. We conclude that KMT2C is a key regulator of ERα activity whose loss uncouples breast cancer proliferation from hormone abundance.

OBJECTIVES: To identify genomic variations in cell-free DNA (cfDNA) and evaluate their clinical utility in small cell lung cancer (SCLC).
MATERIALS AND METHODS: We performed whole genome sequencing using plasma cfDNAs derived from 24 SCLC patients for copy number variation (CNV) analysis, and targeted sequencing using 17 pairs of plasma cfDNA and their matched gDNA for mutation analysis. We defined somatic mutations by comparing cfDNA to its matched gDNA with 5% variant alleles as the cutoff for mutation calls. We applied Kaplan-Meier to correlate the genomic alterations with overall survival (OS) and progression-free survival (PFS).
RESULTS: We observed widespread somatic copy-number alterations and mutations, including amplification of MYC at 8q24, FGF10 at 5p13, SOX2 at 3q26 and FGFR1 at 8p12, as well as deletion of TP53 at 17p13, RASSF1 at 3p21.3, RB1 at 13q14.2, FHIT at 3p14, and PTEN at 10q23. The most frequent mutations were genes involved in chromatin regulation (KMT2D, ARID1A, SETBP1 and PBRM1), PI3K/MTOR pathway(MTOR,PIK13G), Notch1 signalling pathway (NOTCH1), and DNA repair related gene ATRX. Kaplan-Meier analysis revealed poor OS and PFS in patients with somatic mutations in gene SETBP1 (P = 0.0061/0.0264, HR = 4.785/3.841, 95% CI = 2.014-28.25/1.286-16.58) and PBRM1 (P = 0.0276/0.0286, HR = 3.532/3.506, 95% CI = 1.275 to 25.34/1.26-24.87). Poor OS was also associated with somatic mutations in ATRX (P = 0.0099, HR = 4.024, 95% CI = 1.926-42.95), EP300 (P = 0.025/0.0622, HR = 3.382/2.891, 95% CI = 1.448-27.76/1.013-17.29), while poor PFS was associated with ATM mutation (P = 0.0038, HR = 4.604, 95% CI = 2.211-40.93). The mutation index produced by summing up the number of mutations in the five genes was significantly associated with the poor OS/PFS (P = 0.0185/0.0294) after adjusting the effect of the stage.
CONCLUSIONS: Our result supports blood plasma as a promising sample source for the genomic analysis in SCLC patients whose tumor tissues are scarcely available and demonstrates potential clinical utilities of cfDNA-based liquid biopsy for clinical management of this deadly disease.

We report the case of a 27-yr-old male with visual field loss who had a 4.9-cm complex cystic mass in the right occipital lobe. Histologic examination showed pilocytic astrocytoma (PA) with anaplasia, and molecular characterization revealed

BACKGROUND: With advancements in molecular biology techniques, great progress has been made in the understanding of urothelial carcinoma pathogenesis.
OBJECTIVE: To examine the historic description of molecular alterations in bladder cancer and their evolution towards our current comprehension of the biology of the disease.
RESULTS: Historically, a two-pathway model was described from histological and cytogenetic studies: low-grade papillary non-muscle invasive bladder cancers (NMIBC) were described to arise from epithelial hyperplasia with loss of chromosome 9 as an early event, whereas muscle-invasive bladder cancers (MIBC) were considered to develop from dysplasia, associated with genetic instability. Although there could be connections between the 2 pathways, NMIBC and MIBC were largely believed to develop secondary to different molecular alterations. Next-generation sequencing has allowed important insights into cancer biology and a better understanding of the pathways involved in bladder cancer pathogenesis and heterogeneity. Urothelial carcinoma has been found to have a high frequency of somatic mutations compared to other solid tumors, including several mutations in multiple signaling pathways, such as cell cycle regulators (TP53, RB1), RTK/RAS/RAF pathway, PI3K/AKT/mTOR pathway and TERT gene promoter. Epigenetic changes and mutations in chromatin remodeling genes are especially frequent in bladder cancer. Mutations in FGFR3 and KDM6A are more common in NMIBC than in MIBC, whereas mutations in TP53 and KMT2D are more common in MIBC, suggesting the previously hypothesized 2 different pathways, with a subset of tumors progressing from NMIBC to MIBC. Using comprehensive RNA expression profiling studies, at least 5 subtypes of bladder cancer have been identified, the most fundamental division being Basal/Squamous-like and Luminal. These subtypes have different prognoses, natural histories and responses to systemic treatments: Luminal subtypes are enriched with papillary histology and have a better prognosis, while Basal/Squamous-like subtypes are enriched with squamous features, are associated with advanced stage at presentation, and portend a worse prognosis.
CONCLUSION: This new understanding of bladder cancer will optimistically translate into better understanding of this heterogeneous disease and lead to improvement in patient outcome and quality of life through better tailored treatments.

BACKGROUND: Human papillomavirus (HPV)-driven oropharyngeal cancer (OPC) patients are characterised by a better prognosis than their HPV-negative counterparts. However, this significant survival advantage is not homogeneous and among HPV-positive patients those with a smoking history have a significantly increased risk of oncologic failure. The reason why tobacco consumption impacts negatively the prognosis is still elusive. Tobacco might induce additional genetic alterations leading to a more aggressive phenotype. The purpose of this study was to characterise the mutational profile of HPV-positive OPCs by smoking status. We hypothesise a higher frequency of mutations affecting smokers.
METHODS: Targeted next-generation sequencing of 39 genes that are recurrently mutated in head and neck cancers (HNCs) caused by tobacco/alcohol consumption was performed in 62 HPV-driven OPC cases including smokers and non-smokers.
RESULTS: The study population included 37 (60%) non-smokers and 25 (40%) smokers. Twenty (32%) patients had no mutation, 14 (23%) had 1 mutation and 28 (45%) had 2 or more mutations. The most commonly mutated genes regardless of tobacco consumption were PIK3CA (19%), MLL2 (19%), TP53 (8%), FAT 1 (15%), FBXW7 (16%), NOTCH1 (10%) and FGFR3 (10%). Mutation rate was not significantly different in smokers compared with non-smokers even when analyses focused on heavy smokers (>20 pack-years vs. <20 pack-years). Similarly, there was no significant difference in mutations patterns according to tobacco consumption.
CONCLUSION: In HPV-positive patients, smoking does not increase the mutation rate of genes that are recurrently mutated in traditional HNC. Additional studies are warranted to further describe the molecular landscape of HPV-driven OPC according to tobacco consumption.

KDM6A, an X chromosome-encoded histone demethylase and member of the COMPASS-like complex, is frequently mutated in a broad spectrum of malignancies and contributes to oncogenesis with poorly characterized mechanisms. We found that KDM6A loss induced squamous-like, metastatic pancreatic cancer selectively in females through deregulation of the COMPASS-like complex and aberrant activation of super-enhancers regulating ΔNp63, MYC, and RUNX3 oncogenes. This subtype of tumor developed in males had concomitant loss of UTY and KDM6A, suggesting overlapping roles, and points to largely demethylase independent tumor suppressor functions. We also demonstrate that KDM6A-deficient pancreatic cancer is selectively sensitive to BET inhibitors, which reversed squamous differentiation and restrained tumor growth in vivo, highlighting a therapeutic niche for patient tailored therapies.

BACKGROUND: MLL2 has been identified as one of the most frequently mutated genes in a variety of cancers including esophageal squamous cell carcinoma (ESCC). However, its clinical significance and prognostic value in ESCC has not been elucidated. In the present study, we aimed to investigate the expression and role of MLL2 in ESCC.
METHODS: Immunohistochemistry (IHC) and qRT-PCR were used to examine the expression profile of MLL2. Kaplan-Meier survival analysis and univariate and multivariate Cox analyses were used to investigate the clinical and prognostic significance of MLL2 expression in Kazakh ESCC patients. Furthermore, to evaluate the biological function of MLL2 in ESCC, we applied the latest gene editing technique CRISPR/Cas9 to knockout MLL2 in ESCC cell line Eca109. MTT, colony formation, flow cytometry, scratch wound-healing and transwell migration assays were performed to investigate the effect of MLL2 on ESCC cell proliferation and migration. The correlation between MLL2 and epithelial-mesenchymal transition (EMT) was investigated by Western blot assay in vitro and IHC in ESCC tissue, respectively.
RESULTS: Both mRNA and protein expression levels of MLL2 were significantly overexpressed in ESCC patients. High expression of MLL2 was significantly correlated with TNM stage (P = 0.037), tumor differentiation (P = 0.032) and tumor size (P = 0.035). Kaplan-Meier survival analysis showed that patients with low MLL2 expression had a better overall survival than those with high MLL2 expression. Multivariate Cox analysis revealed that lymph node metastasis and tumor differentiation were independent prognostic factors. Knockout of MLL2 in Eca109 inhibited cell proliferation and migration ability, induced cell cycle arrest at G1 stage, but it had no significant effect on apoptosis. In addition, knockout of MLL2 could inhibit EMT by up-regulation of E-Cadherin and Smad7 as well as down-regulation of Vimentin and p-Smad2/3 in ESCC cells. In cancer tissues, the expression of E-Cadherin was negatively correlated with MLL2 expression while Vimentin expression was positively correlated with MLL2 expression.
CONCLUSION: Our results indicate that overexpression of MLL2 predicts poor clinical outcomes and facilitates ESCC tumor progression, and it may exert oncogenic role via activation of EMT. MLL2 may be used as a novel prognostic factor and therapeutic target for ESCC patients.

BACKGROUND: Carotid body tumor (CBT) is a form of head and neck paragangliomas (HNPGLs) arising at the bifurcation of carotid arteries. Paragangliomas are commonly associated with germline and somatic mutations involving at least one of more than thirty causative genes. However, the specific functionality of a number of these genes involved in the formation of paragangliomas has not yet been fully investigated.
METHODS: Exome library preparation was carried out using Nextera® Rapid Capture Exome Kit (Illumina, USA). Sequencing was performed on NextSeq 500 System (Illumina).
RESULTS: Exome analysis of 52 CBTs revealed potential driver mutations (PDMs) in 21 genes: ARNT, BAP1, BRAF, BRCA1, BRCA2, CDKN2A, CSDE1, FGFR3, IDH1, KIF1B, KMT2D, MEN1, RET, SDHA, SDHB, SDHC, SDHD, SETD2, TP53BP1, TP53BP2, and TP53I13. In many samples, more than one PDM was identified. There are also 41% of samples in which we did not identify any PDM; in these cases, the formation of CBT was probably caused by the cumulative effect of several not highly pathogenic mutations. Estimation of average mutation load demonstrated 6-8 mutations per megabase (Mb). Genes with the highest mutation rate were identified.
CONCLUSIONS: Exome analysis of 52 CBTs for the first time revealed the average mutation load for these tumors and also identified potential driver mutations as well as their frequencies and co-occurrence with the other PDMs.

BACKGROUND: Tumour heterogeneity and clonal evolution within a cancer patient are deemed responsible for relapse in malignancies and present challenges to the principles of targeted therapy, for which treatment modality is often decided based on the molecular pathology of the primary tumour. Nevertheless, the clonal architecture in distant relapse of head and neck cancer is fairly unknown.
PATIENTS AND METHODS: For this project, we analysed a cohort of 386 patients within the Austrian Registry of head and neck cancer. We identified 26 patients with material from the primary tumour, the distant metastasis after curative first-line treatment and a germline sample for analysis of clonal evolution. After pathological analyses, these samples were analysed using a targeted massively parallel sequencing (MPS) panel of 257 genes known to be recurrently mutated in head and neck cancer plus a genome-wide SNP-set.
RESULTS: Despite histological diagnosis of distant metastasis, no corresponding mutation in the supposed metastases was found in two of 23 (8.6%) evaluable patients suggesting a primary tumour of the lung instead of a distant metastasis of head and neck cancer. We observed a branched pattern of evolution in 31.6% of the analysed patients. This pattern was associated with a shorter time to distant metastasis, compared with a pattern of punctuated evolution. Structural genomic changes over time were also present in 7 of 12 (60%) evaluable patients with metachronous metastases.
CONCLUSION: Targeted MPS demonstrated substantial heterogeneity at the time of diagnosis and a complex pattern of evolution during disease progression in head and neck cancer. Copy number analyses revealed additional changes that were not detected by mutational analyses. Mutational and structural changes contribute to tumour heterogeneity at diagnosis and progression.

Follicular lymphoma (FL) is presented as a germinal centre B cell lymphoma that is characterized by an indolent clinical course, but remains - paradoxically - largely incurable to date. The last years have seen significant progress in our understanding of FL lymphomagenesis, which is a multi-step process beginning in the bone marrow with the hallmark t(14;18)(q32;q21) translocation. The pathobiology of FL is complex and combines broad somatic changes at the level of both the genome and the epigenome, the latter evidenced by highly recurrent mutations in chromatin-modifying genes such as KMT2D and CREBBP. While the importance of the FL microenvironment has since long been well understood, it has become evident that somatic lesions within tumour cells re-educate normal immune and stromal cells to their advantage. Enhanced understanding of FL pathogenesis is currently leading to refined therapeutic targeting of perturbed biology, paving the way for precision medicine in this lymphoma subtype.