Search

Classic adenoid cystic carcinomas (C-AdCCs) of the breast are rare, relatively indolent forms of triple negative cancers, characterized by recurrent MYB or MYBL1 genetic alterations. Solid and basaloid adenoid cystic carcinoma (SB-AdCC) is considered a rare variant of AdCC yet to be fully characterized. Here, we sought to determine the clinical behavior and repertoire of genetic alterations of SB-AdCCs. Clinicopathologic data were collected on a cohort of 104 breast AdCCs (75 C-AdCCs and 29 SB-AdCCs). MYB expression was assessed by immunohistochemistry and MYB-NFIB and MYBL1 gene rearrangements were investigated by fluorescent in-situ hybridization. AdCCs lacking MYB/MYBL1 rearrangements were subjected to RNA-sequencing. Targeted sequencing data were available for 9 cases. The invasive disease-free survival (IDFS) and overall survival (OS) were assessed in C-AdCC and SB-AdCC. SB-AdCCs have higher histologic grade, and more frequent nodal and distant metastases than C-AdCCs. MYB/MYBL1 rearrangements were significantly less frequent in SB-AdCC than C-AdCC (3/14, 21% vs 17/20, 85% P

While Mek1/2 and Gsk3β inhibition (“2i”) supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female “all-ESC” mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Chromosome 8q gain is associated with poor clinical outcomes in prostate cancer, but the underlying biological mechanisms remain to be clarified. CSN5, a putative androgen receptor (AR) partner that is located on chromosome 8q, is the key subunit of the COP9 signalosome, which deactivates ubiquitin ligases. Deregulation of CSN5 could affect diverse cellular functions that contribute to tumor development, but there has been no comprehensive study of its function in prostate cancer. The clinical significance of CSN5 amplification/overexpression was evaluated in 16 prostate cancer clinical cohorts. Its oncogenic activity was assessed by genetic and pharmacologic perturbations of CSN5 activity in prostate cancer cell lines. The molecular mechanisms of CSN5 function were assessed, as was the efficacy of the CSN5 inhibitor CSN5i-3 in vitro and in vivo. Finally, the transcription cofactor activity of CSN5 in prostate cancer cells was determined. The prognostic significance of CSN5 amplification and overexpression in prostate cancer was independent of MYC amplification. Inhibition of CSN5 inhibited its oncogenic function by targeting AR signaling, DNA repair, multiple oncogenic pathways, and spliceosome regulation. Furthermore, inhibition of CSN5 repressed metabolic pathways, including oxidative phosphorylation and glycolysis in AR-negative prostate cancer cells. Targeting CSN5 with CSN5i-3 showed potent antitumor activity in vitro and in vivo. Importantly, CSN5i-3 synergizes with PARP inhibitors to inhibit prostate cancer cell growth. CSN5 functions as a transcription cofactor to cooperate with multiple transcription factors in prostate cancer. Inhibiting CSN5 strongly attenuates prostate cancer progression and could enhance PARP inhibition efficacy in the treatment of prostate cancer.

Karyotyping remains an invaluable tool to researchers exploring the cause and consequence of genomic instability in biologic systems. It allows investigators to survey the entire chromosome complement in individual cells and in a single experiment, visualize, and measure different forms or features of instability such as aneuploidy, ongoing chromosomal instability, DNA damage/mis-repair, telomere erosion, chromosome mis-segregation, or defects in cell cycle progression. This chapter describes the combined use of conventional (DAPI-banding) and spectral karyotyping (SKY) to characterize genomic instability in murine cerebellar granule neuron progenitors (CGNPs), using CGNPs with conditional deletion of Atr as a positive control for chromosomal rearrangements. Protocols for preparing slides (metaphase spreads) from fixed cell suspension, DAPI-banding, and spectral karyotyping (SKY) are included. Pertinent aspects of image acquisition and analysis are detailed. These protocols can likely be adapted to other tissue types (murine or human).

Molecular characterization of adrenocortical carcinomas (ACC) by The Cancer Genome Atlas (TCGA) has highlighted a high prevalence of TERT alterations, which are associated with disease progression. Herein, 78 ACC were profiled using a combination of next generation sequencing (n = 76) and FISH (n = 9) to assess for TERT alterations. This data was combined with TCGA dataset (n = 91). A subset of borderline adrenocortical tumors (n = 5) and adrenocortical adenomas (n = 7) were also evaluated. The most common alteration involving the TERT gene involved gains/amplifications, seen in 22.2% (37/167) of cases. In contrast, “hotspot” promoter mutations (C > T promoter mutation at position -124, 7/167 cases, 4.2%) and promoter rearrangements (2/165, 1.2%) were rare. Recurrent co-alterations included 22q copy number losses seen in 24% (9/38) of cases. Although no significant differences were identified in cases with and without TERT alterations pertaining to age at presentation, tumor size, weight, laterality, mitotic index and Ki67 labeling, cases with TERT alterations showed worse outcomes. Metastatic behavior was seen in 70% (28/40) of cases with TERT alterations compared to 51.2% (65/127, p = 0.04) of cases that lacked these alterations. Two (of 5) borderline tumors showed amplifications and no TERT alterations were identified in 7 adenomas. In the borderline group, 0 (of 4) patients with available follow up had adverse outcomes. We found that TERT alterations in ACC predominantly involve gene amplifications, with a smaller subset harboring “hotspot” promoter mutations and rearrangements, and 70% of TERT-altered tumors are associated with metastases. Prospective studies are needed to validate the prognostic impact of these findings.

Lipids play vital roles in cellular homeostasis and regulate pluripotency of human stem cells. However, the impact of lipids on murine pluripotent stem cells is unclear. While Mek1/2 and Gsk3β inhibition (“2i”) supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of ZCAN4 and DNMT3s that prevent telomere shortening and DNA hypomethylation. In concert with 2i, lipids induce a formative-like pluripotent state through direct stimulation of Mek-mediated Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female “all-ESC” mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Purpose: Previous sequencing studies revealed that alterations of genes associated with DNA damage response (DDR) are enriched in men with metastatic castration-resistant prostate cancer (mCRPC). BRCA2, a DDR and cancer susceptibility gene, is frequently deleted (homozygous and heterozygous) in men with aggressive prostate cancer. Here we show that patients with prostate cancer who have lost a copy of BRCA2 frequently lose a copy of tumor suppressor gene RB1; importantly, for the first time, we demonstrate that co-loss of both genes in early prostate cancer is sufficient to induce a distinct biology that is likely associated with worse prognosis. Experimental Design: We prospectively investigated underlying molecular mechanisms and genomic consequences of co-loss of BRCA2 and RB1 in prostate cancer. We used CRISPR-Cas9 and RNAi-based methods to eliminate these two genes in prostate cancer cell lines and subjected them to in vitro studies and transcriptomic analyses. We developed a 3-color FISH assay to detect genomic deletions of BRCA2 and RB1 in prostate cancer cells and patient-derived mCRPC organoids. Results: In human prostate cancer cell lines (LNCaP and LAPC4), loss of BRCA2 leads to the castration-resistant phenotype. Co-loss of BRCA2-RB1 in human prostate cancer cells induces an epithelial-to-mesenchymal transition, which is associated with invasiveness and a more aggressive disease phenotype. Importantly, PARP inhibitors attenuate cell growth in human mCRPC-derived organoids and human CRPC cells harboring single-copy loss of both genes. Conclusions: Our findings suggest that early identification of this aggressive form of prostate cancer offers potential for improved outcomes with early introduction of PARP inhibitor–based therapy. See related commentary by Mandigo and Knudsen, p. 1784

BACKGROUND: Molecular and immunohistochemistry-based profiling of prostatic adenocarcinoma has revealed frequent Androgen Receptor (AR) gene and protein alterations in metastatic disease. This includes an AR-null non-neuroendocrine phenotype of metastatic castrate resistant prostate cancer which may be less sensitive to androgen receptor signaling inhibitors. This AR-null non-neuroendocrine phenotype is thought to be associated with TP53 and RB1 alterations. Herein, we have correlated molecular profiling of metastatic castrate resistant prostate cancer with AR/P53/RB immunohistochemistry and relevant clinical correlates. DESIGN: Twenty-seven cases of metastatic castrate resistant prostate cancer were evaluated using histopathologic examination to rule out neuroendocrine differentiation. A combination of a hybridization exon-capture next-generation sequencing-based assay (n = 26), fluorescence in situ hybridization for AR copy number status (n = 16), and immunohistochemistry for AR (n = 27), P53 (n = 24) and RB (n = 25) was used to profile these cases. RESULTS: Of 27 metastatic castrate resistant prostate cancer cases, 17 had AR amplification and showed positive nuclear expression of AR by immunohistochemistry. Nine cases lacked AR copy number alterations using next-generation sequencing/fluorescence in situ hybridization. A subset of these metastatic castrate resistant prostate cancer cases demonstrated the AR-null phenotype by immunohistochemistry (five cases and one additional case where next-generation sequencing failed). Common co-alterations in these cases involved the TP53, RB1, and PTEN genes and all these patients received prior therapy with androgen receptor signaling inhibitors (abiraterone and/or enzalutamide). CONCLUSIONS: Our study suggests that AR immunohistochemistry may distinguish AR-null from AR-expressing cases in the metastatic setting. AR-null status informs clinical decision-making regarding continuation of therapy with androgen receptor signaling inhibitors and consideration of other treatment options. This might be a relevant and cost-effective diagnostic strategy when there is limited access and/or limited tumor material for molecular testing.

Hurthle cell carcinomas (HCCs) are refractory to radioactive iodine and unresponsive to chemotherapeutic agents, with a fatality rate that is the highest among all types of thyroid cancer after anaplastic thyroid cancer. Our previous study on the genomic landscape of HCCs identified a high incidence of disruptions of mTOR pathway effectors. Here, we report a detailed analysis of mTOR signaling in cell line and patient-derived xenograft mouse models of HCCs. We show that mTOR signaling is upregulated and that targeting mTOR signaling using mTOR inhibitors suppresses tumor growth in primary tumors and distant metastasis. Mechanistically, ablation of mTOR signaling impaired the expression of p-S6 and cyclin A2, resulting in the decrease of the S phase and blocking of cancer cell proliferation. Strikingly, mTOR inhibitor treatment significantly reduced lung metastatic lesions, with the decreased expression of Snail in xenograft tumors. Our data demonstrate that mTOR pathway blockade represents a novel treatment strategy for HCC.

Background EGFR amplification occurs in about 1% of metastatic colorectal cancers (mCRCs) but is not routinely tested as a prognostic or predictive biomarker for patients treated with anti-EGFR monoclonal antibodies. Herein, we aimed to characterize the clinical and molecular landscape of EGFR-amplified mCRC. Methods In this multinational cohort study, we compared clinical data of 62 patients with EGFR-amplified vs 1459 EGFR nonamplified mCRC, as well as comprehensive genomic data of 35 EGFR-amplified vs 439 EGFR nonamplified RAS/BRAF wild-type and microsatellite stable (MSS) tumor samples. All statistical tests were 2-sided. Results EGFR amplification was statistically significantly associated with left primary tumor sidedness and RAS/BRAF wild-type status. All EGFR-amplified tumors were MSS and HER2 nonamplified. Overall, EGFR-amplified samples had higher median fraction of genome altered compared with EGFR-nonamplified, RAS/BRAF wild-type MSS cohort. Patients with EGFR-amplified tumors reported longer overall survival (OS) (median OS = 71.3 months, 95% confidence interval [CI] = 50.7 to not available [NA]) vs EGFR-nonamplified ones (24.0 months; 95% CI = 22.8 to 25.6; hazard ratio [HR] = 0.30, 95% CI = 0.20 to 0.44; P < .001; adjusted HR = 0.46, 95% CI = 0.30 to 0.69; P < .001). In the subgroup of patients with RAS/BRAF wild-type mCRC exposed to anti-EGFR-based therapy, EGFR amplification was again associated with better OS (median OS = 54.0 months, 95% CI = 35.2 to NA, vs 29.1 months, 95% CI = 27.0 to 31.9, respectively; HR = 0.46, 95% CI = 0.28 to 0.76; P = .002). Conclusion Patients with EGFR-amplified mCRC represent a biologically defined subgroup and merit dedicated clinical trials with novel and more potent EGFR-targeting strategies beyond single-agent monoclonal antibodies.

Renal medullary carcinoma is a rare but highly aggressive type of renal cancer occurring in patients with sickle cell trait or rarely with other hemoglobinopathies. Loss of SMARCB1 protein expression, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, has emerged as a key diagnostic feature of these tumors. However, the molecular mechanism underlying this loss remains unclear. We retrospectively identified 20 patients diagnosed with renal medullary carcinoma at two institutions from 1996 to 2017. All patients were confirmed to have sickle cell trait, and all tumors exhibited a loss of SMARCB1 protein expression by immunohistochemistry. The status of SMARCB1 locus was examined by fluorescence in situ hybridization (FISH) using 3-color probes, and somatic alterations were detected by targeted next-generation sequencing platforms. FISH analysis of all 20 cases revealed 11 (55%) with concurrent hemizygous loss and translocation of SMARCB1, 6 (30%) with homozygous loss of SMARCB1, and 3 (15%) without structural or copy number alterations of SMARCB1 despite protein loss. Targeted sequencing revealed a pathogenic somatic mutation of SMARCB1 in one of these 3 cases that were negative by FISH. Tumors in the 3 subsets with different FISH findings largely exhibited similar clinicopathologic features, however, homozygous SMARCB1 deletion was found to show a significant association with the solid growth pattern, whereas tumors dominated by reticular/cribriform growth were enriched for SMARCB1 translocation. Taken together, we demonstrate that different molecular mechanisms underlie the loss of SMARCB1 expression in renal medullary carcinoma. Biallelic inactivation of SMARCB1 occurs in a large majority of cases either via concurrent hemizygous loss and translocation disrupting SMARCB1 or by homozygous loss.

Summary: The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a “normal” human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening. : To address limitations with current preclinical glioblastoma (GBM) models, Linkous et al. establish a “GLICO” (cerebral organoid glioma) model to retro-engineer patient-specific GBMs using patient-derived glioma stem cells and human cerebral organoids. Resulting tumors closely phenocopy patient GBMs and are supported by tumor microtubes that promote invasion into host tissue. Keywords: cerebral organoids, glioma stem cells, glioblastoma, glioma, tumor microtubes, human embryonic stem cells, brain tumors, stem-cell-based disease models, tissue engineering, cancer stem cells

OBJECTIVES: DAXX immunohistochemistry (IHC) is often used as a surrogate for sequencing. We aimed to elucidate the sensitivity of IHC for DAXX mutation. METHODS: All pancreatic neuroendocrine tumors (PanNETs) with DAXX mutations detected by sequencing and a subset of DAXX wild type PanNETs were analyzed for DAXX expression by IHC. RESULTS: Of 154 PanNETs with MSK-IMPACT testing, 36 (30%) harbored DAXX mutations. DAXX mutations were associated with TSC2 mutations (46% vs. 10%, P < 0.0001), tended to co-occur with MEN1 mutations (63% vs 49%, P = 0.11), and tended to be mutually exclusive with ATRX mutations (11% vs 25%, P = 0.053). Of 27 available DAXX mutant PanNETs, 23 lost DAXX expression (85.2%). All 4 DAXX mutants with retained expression harbored DAXX mutations within the SUMO-interacting motif of the last exon. Telomere-specific fluorescence in situ hybridization demonstrated alternative lengthening of telomeres in all 4 cases. Of 20 PanNETs with wild type DAXX, 19 retained DAXX IHC expression (95%). CONCLUSIONS: The sensitivity and specificity of IHC for DAXX mutation is 85% and 95%, respectively. Last exon DAXX mutant PanNETs often show alternative lengthening of telomeres despite retained DAXX expression, likely due to escape of non-mediated decay.

SMARCB1-deficient sinonasal carcinoma (SNC) is an aggressive malignancy characterized by INI1 loss mostly owing to homozygous SMARCB1 deletion. With the exception of a few reported cases, these tumors have not been thoroughly studied by massive parallel sequencing (MPS). A retrospective cohort of 22 SMARCB1-deficient SNCs were studied by light microscopy, immunohistochemistry, fluorescence in situ hybridization (n = 9), targeted exome MPS (n = 12), and Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) (n = 10), a bioinformatics pipeline for copy number/zygosity assessment. SMARCB1-deficient SNC was found in 13 (59%) men and 9 (41%) women. Most common growth patterns were the basaloid pattern (59%), occurring mostly in men (77%), and plasmacytoid/eosinophilic/rhabdoid pattern (23%), arising mostly in women (80%). The former group was significantly younger (median age = 46 years, range = 24-54, vs 79 years, range = 66-95, p 0.0001). Clear cell, pseudoglandular, glandular, spindle cell, and sarcomatoid features were variably present. SMARCB1-deficient SNC expressed cytokeratin (100%), p63 (72%), neuroendocrine markers (52%), CDX-2 (44%), S-100 (25%), CEA (4/4 cases), Hepatocyte (2/2 cases), and aberrant nuclear β-catenin (1/1 case). SMARCB1 showed homozygous deletion (68%), hemizygous deletion (16%), or truncating mutations associated with copy neutral loss of heterozygosity (11%). Coexisting genetic alterations were 22q loss including loss of NF2 and CHEK2 (50%), chromosome 7 gain (25%), and TP53 V157F, CDKN2A W110∗, and CTNNB1 S45F mutations. At 2 years and 5 years, the disease-specific survival and disease-free survival were 70% and 35% and 13% and 0%, respectively. SMARCB1-deficient SNCs are phenotypically and genetically diverse, and these distinctions warrant further investigation for their biological and clinical significance.

Primary pleomorphic adenomas (PAs) and mucoepidermoid carcinomas (MECs) of the breast are vanishingly rare. Here we sought to determine whether breast PAs and MECs would be underpinned by the fusion genes reported to occur in their salivary gland counterparts. Our study included three breast PAs and one breast MEC, which were subjected to RNA sequencing (PAs, n = 2; MEC, n = 1) or to Archer FusionPlex sequencing (PA, n = 1). Our analyses revealed the presence of the HMGA2-WIF1 fusion gene in breast PA3, the CTNNB1-PLAG1 fusion gene in breast PA2, and the CRTC1-MAML2 fusion gene in the breast MEC analyzed (1/1). No oncogenic fusion genes were detected in breast PA1, and no additional oncogenic fusion genes were detected in the cases studied. The presence of the fusion genes identified was validated by fluorescence in situ hybridization (n = 1), reverse transcription-PCR (n = 1), or by both methods (n = 1). Taken together, our findings indicate that PAs and MECs arising in the breast resemble their salivary gland counterparts not only phenotypically but also at the genetic level. Furthermore, our data suggest that the molecular analysis of breast PAs and MECs might constitute a useful tool to aid in their differential diagnosis.

Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse., eLife digest Cells in the body remain healthy by tightly preventing and repairing random changes, or mutations, in their genetic material. In cancer cells, however, these mechanisms can break down. When these cells grow and multiply, they can then go on to accumulate many mutations. As a result, cancer cells in the same tumor can each contain a unique combination of genetic changes. This genetic heterogeneity has the potential to affect how cancer responds to treatment, and is increasingly becoming appreciated clinically. For example, if a drug only works against cancer cells carrying a specific mutation, any cells lacking this genetic change will keep growing and cause a relapse. However, it is still difficult to quantify and understand genetic heterogeneity in cancer. Copy number alterations (or CNAs) are a class of mutation where large and small sections of genetic material are gained or lost. This can result in cells that have an abnormal number of copies of the genes in these sections. Here, Baslan et al. set out to explore how CNAs might vary between individual cancer cells within the same tumor. To do so, thousands of individual cancer cells were isolated from human breast tumors, and a technique called single-cell genome sequencing used to screen the genetic information of each of them. These experiments confirmed that CNAs did differ – sometimes dramatically – between patients and among cells taken from the same tumor. For example, many of the cells carried extra copies of well-known cancer genes important for treatment, but the exact number of copies varied between cells. This heterogeneity existed for individual genes as well as larger stretches of DNA: this was the case, for instance, for an entire section of chromosome 8, a region often affected in breast and other tumors. The work by Baslan et al. captures the sheer extent of genetic heterogeneity in cancer and in doing so, highlights the power of single-cell genome sequencing. In the future, a finer understanding of the genetic changes present at the level of an individual cancer cell may help clinicians to manage the disease more effectively.

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer. Iacobuzio-Donahue and colleagues use integrated transcriptomic, histologic and mutational data to analyze squamous features of pancreatic ductal adenocarcinoma (PDAC), further refining the understanding of heterogeneity and evolution in PDAC.

Micropapillary urothelial carcinoma (MPUC) is a rare but an aggressive variant of urothelial carcinoma. MPUC has been shown to commonly exhibit ERBB2 amplification and HER2 protein overexpression, but the frequency and distribution of these findings within micropapillary (MP) and not otherwise specified (NOS) components of tumors with mixed histology have not been addressed. Therefore, we evaluated ERBB2 amplification and HER2 expression in 43 MPUC cases by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Of the 35 tumors containing both MP and NOS components, ERBB2 amplification was present in both the MP and NOS components of 12 tumors (34.3%), in only the MP component of 11 tumors (31.4%), and exclusively in the NOS component of 4 tumors (11.4%). HER2 protein overexpression was significantly more commonly present in the MP component compared to the NOS component within the same tumor (68.6% versus 34.3%, P = .012). Overall, there was a moderately positive correlation between HER2 protein expression and ERBB2 amplification in both MP (ρ = 0.59, P < .001) and NOS (ρ = 0.70, P < .001) components. All MP/NOS areas with IHC score 3+ and none of MP/NOS areas with IHC score 0 were associated with ERBB2 amplification. We conclude that ERBB2 amplification and HER2 overexpression are preferentially but not exclusively identified in the MP component compared to the NOS component within the same tumor. Our findings identify the presence of intratumoral heterogeneity of ERBB2 amplification and HER2 expression in MPUC and provide grounds for further investigation into the mechanisms underlying the development of MPUC.

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.

Background Renal medullary carcinoma (RMC) is a rare and aggressive type of kidney cancer that primarily affects young adults with sickle cell trait; outcomes are poor despite treatment. Identifying molecular features of this tumor could provide biologic rationale for novel targeted therapies. The objective was to report on clinical outcomes with systemic therapy and characterize molecular features. Patients and Methods This was a retrospective analysis on 36 patients given a pathologic diagnosis of RMC at one institution from 1995 to 2015. Tumors were analyzed for expression of SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily B, Member 1 (SMARCB1) through immunohistochemistry and for genomic alterations with fluorescence in situ hybridization for SMARCB1, and targeted next-generation sequencing. Time from initiation of therapy to progression of disease and overall survival were calculated using the Kaplan–Meier method. Results The median age in the cohort was 28 (range, 12-72) years, and all patients tested had sickle cell trait. Overall survival was 5.8 months (95% confidence interval [CI], 4.1-10.9) and for 12 patients who received platinum-based therapy, median progression-free survival was 2.5 months (95% CI, 1.2-not reached). A total of 10 available tumors underwent analysis with fluorescence in situ hybridization for SMARCB1; this revealed loss of heterozygosity with concurrent translocation in 8, and biallelic loss in 2. Next-generation targeted sequencing showed no recurring mutations. Conclusions Outcome was generally poor in this cohort of patients with RMC. Uniform loss of SMARCB1 is a key molecular feature in this tumor and mechanism of loss appears to be mostly through translocations and deletions.

Summary: Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis. : In a human neural stem cell model of low-grade astrocytoma, Modrek et al. show that mutant IDH1 and loss of P53 and ATRX together block differentiation via disassociation of SOX2 from putative enhancers. This occurs because of disruption of chromatin looping secondary to hypermethylation at CTCF motifs. Keywords: low-grade glioma, astrocytoma, IDH, P53, ATRX, neural stem cells, SOX2, chromatin looping, CTCF, DNA methylation

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

Aims Secretory carcinoma of the breast (SCB) is a rare histological type of breast carcinoma with a generally indolent clinical behaviour. We aim to elucidate the clinical, pathological and molecular findings of SCB cases and identify characteristics associated with aggressive clinical courses. Methods and results Fourteen patients with SCB were identified, including 12 women and two men, with a median age of 56 years (range = 8-81 years). Clinical data, histological diagnosis, molecular findings and follow-up were reviewed. Eight patients presented with palpable masses and four patients with radiographic abnormalities. All cases were unilateral. Surgical procedures included excisional biopsies and ipsilateral mastectomies. In 10 cases, oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) results were obtained, with six cases positive for ER and three positive for PR. All cases lacked HER2 overexpression. Sentinel lymph node biopsy was performed in 10 cases, and two patients had axillary lymph node metastasis. Follow-up ranged from 21 to 212 months (median = 70 months). Two patients developed distant metastasis of SCB. Molecular analysis of these aggressive tumours revealed amplification of the 16p13.3 locus, a TERT promotor mutation and loss of 9p21.3 locus. Review of the literature for SCB cases with distant metastasis was performed. Conclusions Although SCBs are generally associated with a favourable prognosis, our study and review demonstrate that a subset of SCBs may develop distant metastases. Further studies are warranted to identify markers predictive of more aggressive clinical behaviour in this rare breast cancer subtype.

SummaryRecent studies indicate that pancreatic cancer expression profiles are variable and largely reflect a classical or basal-type phenotype. We performed genetic sequencing, RNA-seq, and histologic review of multiregion sampled pancreatic cancers and found that squamous and squamoid features, indicators of poor prognosis, correlate with a “basal-like” expressional type. Cancers with squamous features were more likely to have truncal mutations in chromatin modifier genes and intercellular heterogeneity for MYC amplification that was associated with entosis. In most patients the basal phenotype coexisted with a glandular component, and phylogenetic studies indicated that it arose from a subclonal population in the tumor. These data provide a unifying paradigm for understanding the interrelationship of basal-type features, squamous histology, and somatic mutations in chromatin modifier genes in the context of the clonal evolution of pancreatic cancer.

Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. We report that ATR, which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. Our data show that p53-driven apoptosis and cell cycle arrest – and, in the absence of p53, non-apoptotic cell death – redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition.

Renal cell carcinomas with unclassified histology (uRCC) constitute a significant portion of aggressive non-clear cell renal cell carcinomas that have no standard therapy. The oncogenic drivers in these tumours are unknown. Here we perform a molecular analysis of 62 high-grade primary uRCC, incorporating targeted cancer gene sequencing, RNA sequencing, single-nucleotide polymorphism array, fluorescence in situ hybridization, immunohistochemistry and cell-based assays. We identify recurrent somatic mutations in 29 genes, including NF2 (18%), SETD2 (18%), BAP1 (13%), KMT2C (10%) and MTOR (8%). Integrated analysis reveals a subset of 26% uRCC characterized by NF2 loss, dysregulated Hippo–YAP pathway and worse survival, whereas 21% uRCC with mutations of MTOR, TSC1, TSC2 or PTEN and hyperactive mTORC1 signalling are associated with better clinical outcome. FH deficiency (6%), chromatin/DNA damage regulator mutations (21%) and ALK translocation (2%) distinguish additional cases. Altogether, this study reveals distinct molecular subsets for 76% of our uRCC cohort, which could have diagnostic and therapeutic implications., A subset of renal cell carcinomas have uncertain histology and are aggressive in nature. Here, the authors examine this group of unclassified renal cancers using genomics techniques and identify further subclasses of the tumours that have differing prognoses.

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2. Heterogeneous 89Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2-amplified EG cancer. Significance: Analysis of patients with ERBB2-amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR/ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones. See related commentary by Klempner and Catenacci, p. 166. This article is highlighted in the In This Issue feature, p. 151

The molecular foundations of Hürthle cell carcinoma (HCC) are poorly understood. Here, we describe a comprehensive genomic characterization of 56 primary HCC tumors that span the spectrum of tumor behavior. We elucidate the mutational profile and driver mutations and show that they exhibit a wide range of recurrent mutations. Notably, we report an extremely high number of disruptive mutations to both protein-coding and tRNA-encoding regions of the mitochondrial genome. We reveal unique chromosomal landscapes that involve whole-chromosomal duplications of chromosomes 5 and 7 and widespread loss of heterozygosity arising from haploidization and copy number–neutral uniparental disomy. We also identify fusion genes and disrupted signaling pathways that may drive disease pathogenesis.

Significance BCL2 selective inhibitors are promising agents currently under clinical investigation for treatment of BCL2-dependent cancers. However, the clinical activity of BCL2 inhibitors in patients with diffuse large B cell lymphoma (DLBCL) has been disappointing. In this study, we identified PMAIP1/NOXA gene amplification as a marker of sensitivity to BCL2 inhibitors in DLBCL. Cells lacking NOXA amplification were less sensitive to BCL2 inhibitors due to codependency on MCL1 and BCL2 proteins. We show that pharmacologic induction of NOXA by the HDAC inhibitor panobinostat primes DLBCL to BCL2 inhibitor-induced cell death by disrupting the codependency on BCL2 and MCL1, mimicking the biologic effects of NOXA genetic amplification. Our data provide a mechanistic rationale for combining HDAC inhibitors with BCL2 inhibitors in DLBCL., Although diffuse large B cell lymphoma (DLBCL) cells widely express the BCL2 protein, they rarely respond to treatment with BCL2-selective inhibitors. Here we show that DLBCL cells harboring PMAIP1/NOXA gene amplification were highly sensitive to BCL2 small-molecule inhibitors. In these cells, BCL2 inhibition induced cell death by activating caspase 9, which was further amplified by caspase-dependent cleavage and depletion of MCL1. In DLBCL cells lacking NOXA amplification, BCL2 inhibition was associated with an increase in MCL1 protein abundance in a BIM-dependent manner, causing a decreased antilymphoma efficacy. In these cells, dual inhibition of MCL1 and BCL2 was required for enhanced killing. Pharmacologic induction of NOXA, using the histone deacetylase inhibitor panobinostat, decreased MCL1 protein abundance and increased lymphoma cell vulnerability to BCL2 inhibitors in vitro and in vivo. Our data provide a mechanistic rationale for combination strategies to disrupt lymphoma cell codependency on BCL2 and MCL1 proteins in DLBCL.

Copy-number alterations (CNA) are among the most common molecular events in human prostate cancer genomes and are associated with worse prognosis. Identification of the oncogenic drivers within these CNAs is challenging due to the broad nature of these genomic gains or losses which can include large numbers of genes within a given region. Here, we profiled the genomes of four genetically engineered mouse prostate cancer models that reflect oncogenic events common in human prostate tumors, with the goal of integrating these data with human prostate cancer datasets to identify shared molecular events. Met was amplified in 67% of prostate tumors from Pten p53 prostate conditional null mice and in approximately 30% of metastatic human prostate cancer specimens, often in association with loss of PTEN and TP53. In murine tumors with Met amplification, Met copy-number gain and expression was present in some cells but not others, revealing intratumoral heterogeneity. Forced MET overexpression in non–MET-amplified prostate tumor cells activated PI3K and MAPK signaling and promoted cell proliferation and tumor growth, whereas MET kinase inhibition selectively impaired the growth of tumors with Met amplification. However, the impact of MET inhibitor therapy was compromised by the persistent growth of non–Met-amplified cells within Met-amplified tumors. These findings establish the importance of MET in prostate cancer progression but reveal potential limitations in the clinical use of MET inhibitors in late-stage prostate cancer. Mol Cancer Ther; 14(1); 278–88. ©2014 AACR.

Myoepithelial carcinoma (MECA) is an aggressive salivary gland cancer with largely unknown genetic features. Here we comprehensively analyze molecular alterations in 40 MECAs using integrated genomic analyses. We identify a low mutational load, and high prevalence (70%) of oncogenic gene fusions. Most fusions involve the PLAG1 oncogene, which is associated with PLAG1 overexpression. We find FGFR1-PLAG1 in seven (18%) cases, and the novel TGFBR3-PLAG1 fusion in six (15%) cases. TGFBR3-PLAG1 promotes a tumorigenic phenotype in vitro, and is absent in 723 other salivary gland tumors. Other novel PLAG1 fusions include ND4-PLAG1; a fusion between mitochondrial and nuclear DNA. We also identify higher number of copy number alterations as a risk factor for recurrence, independent of tumor stage at diagnosis. Our findings indicate that MECA is a fusion-driven disease, nominate TGFBR3-PLAG1 as a hallmark of MECA, and provide a framework for future diagnostic and therapeutic research in this lethal cancer., Myoepithelial carcinoma (MECA) is a rare aggressive salivary gland cancer. Here, the authors analyze the genomic landscape of MECA and identify a high prevalence of oncogenic gene fusions, primarily PLAG1 fusions, highlighting TGFBR3-PLAG1 as a potential hallmark of MECA.

Chromophobe renal cell carcinoma (chRCC) typically shows ~7 chromosome losses (1, 2, 6, 10, 13, 17, and 21) and ~31 exonic somatic mutations, yet carries ~5%-10% metastatic incidence. Since extensive chromosomal losses can generate proteotoxic stress and compromise cellular proliferation, it is intriguing how chRCC, a tumor with extensive chromosome losses and a low number of somatic mutations, can develop lethal metastases. Genomic features distinguishing metastatic from nonmetastatic chRCC are unknown. An integrated approach, including whole-genome sequencing (WGS), targeted ultradeep cancer gene sequencing, and chromosome analyses (FACETS, OncoScan, and FISH), was performed on 79 chRCC patients including 38 metastatic (M-chRCC) cases. We demonstrate that TP53 mutations (58%), PTEN mutations (24%), and imbalanced chromosome duplication (ICD, duplication of ≥ 3 chromosomes) (25%) were enriched in M-chRCC. Reconstruction of the subclonal composition of paired primary-metastatic chRCC tumors supports the role of TP53, PTEN, and ICD in metastatic evolution. Finally, the presence of these 3 genomic features in primary tumors of both The Cancer Genome Atlas kidney chromophobe (KICH) (n = 64) and M-chRCC (n = 35) cohorts was associated with worse survival. In summary, our study provides genomic insights into the metastatic progression of chRCC and identifies TP53 mutations, PTEN mutations, and ICD as high-risk features.

Myoepithelial carcinoma (MECA) is an aggressive type of salivary gland cancer with largely unknown molecular features. MECA may arise de novo or result from oncogenic transformation of a pre-existing pleomorphic adenoma (MECA ex-PA). We comprehensively analyzed the molecular alterations in MECA with integrated genomic analyses. We identified a low mutational load (0.5/MB), but a high prevalence of fusion oncogenes (28/40 tumors; 70%). We foundFGFR1-PLAG1in 7 (18%) cases, and the novelTGFBR3-PLAG1fusion in 6 (15%) cases.TGFBR3-PLAG1was specific for MECA de novo tumors or the malignant component of MECA ex-PA, was absent in 723 other salivary gland tumors, and promoted a tumorigenic phenotype in vitro. We discovered other novelPLAG1fusions, includingND4-PLAG1,which is an oncogenic fusion between mitochondrial and nuclear DNA. One tumor harbored anMSN-ALKfusion, which was tumorigenic in vitro, and targetable with ALK inhibitors. Certain gene fusions were predicted to result in neoantigens with high MHC binding affinity. A high number of copy number alterations was associated with poorer prognosis. Our findings indicate that MECA is a fusion-driven disease, nominateTGFBR3-PLAG1as a hallmark of MECA, and provide a framework for future steps of diagnostic and therapeutic research in this lethal cancer.

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAF

Objectives To improve the overall accuracy of diagnosis in needle biopsies of renal masses, especially small renal masses (SRMs), using fluorescence in situ hybridization (FISH), and to develop a renal cortical neoplasm classification decision tree based on genomic alterations detected by FISH. Patients and Methods Ex vivo fine needle aspiration biopsies of 122 resected renal cortical neoplasms were subjected to FISH using a series of seven-probe sets to assess gain or loss of 10 chromosomes and rearrangement of the 11q13 locus. Using specimen (nephrectomy)-histology as the ‘gold standard’, a genomic aberration-based decision tree was generated to classify specimens. The diagnostic potential of the decision tree was assessed by comparing the FISH-based classification and biopsy histology with specimen histology. Results Of the 114 biopsies diagnostic by either method, a higher diagnostic yield was achieved by FISH (92 and 96%) than histology alone (82 and 84%) in the 65 biopsies from SRMs (

Array comparative genomic hybridization (aCGH) has yet to be fully leveraged in a prognostic setting in chronic lymphocytic leukemia (CLL). Genomic imbalance was assessed in 288 CLL specimens using a targeted array. Based on 20 aberrations in a hierarchical manner, all 228 treatment-naive specimens were classified into a group with poor outcome (20.6%) exhibiting at least one aberration that was univariately associated with adverse outcome (gain: 2p, 3q, 8q, 17q, loss: 7q, 8p, 11q, 17p, 18p), good outcome (32.5%) showing 13q14 loss without any of the other 10 aberrations (gain: 1p, 7p, 12, 18p, 18q, 19, loss: 4p, 5p, 6q, 7p) or intermediate outcome (remainder). The three groups were significantly separated with respect to time to first treatment and overall survival (p < 0.001), and validation of the stratification scheme was performed in two independent datasets. Gain of 3q and 8q, and 17p loss were determined to be independent unfavorable prognostic biomarkers. TP53, NOTCH1 and SF3B1 mutations correlated with the presence of one poor outcome aCGH marker, at a considerably higher frequency than when only considering poor risk aberrations routinely detected by fluorescence in situ hybridization (FISH). These data support genomic imbalance evaluation in CLL by aCGH to assist in risk stratification.

Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors.

Conventional cytogenetics in conjunction with Fluorescence in Situ Hybridization (FISH) continues to remain an important and integral component in the diagnosis and management of solid tumors. The ability to effectively detect the vast majority of clinically relevant chromosomal aberrations with a rapid-to-acceptable turnaround time makes them the most cost-effective screening/detection tool currently available in modern pathology. In this review, we describe a representative set of solid tumors in which chromosomal analysis and/or FISH plays a significant role in the routine clinical management of solid tumors.

Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.

Background: Hurthle cell carcinoma (HCC) is an enigmatic malignancy of the thyroid that can behave in an aggressive fashion, sometimes lethal, yet its molecular foundations are poorly understood. Some HCC have a good prognosis (minimally invasive, HMIN) whereas others can be extremely aggressive (widely invasive, HWIDE), leading to metastasis and death. HCCs were not included in the TCGA thyroid cancer study, which focused solely on papillary thyroid carcinomas. To understand the development of HCC and unveil its molecular mechanism, we performed a comprehensive genomic characterization of 56 primary HCCs that span the spectrum of tumor behavior and investigated the role of TMEM233/PRKAB1 fusion as a critical driver of oncogenesis in HCC. Methods: Tumor and matched normal specimens were obtained from 56 patients with primary HCC. Tumors were classified into minimally invasive ( n=24) or widely invasive subtype (n=32). Whole exome sequencing was used to identify somatic mutations. Copy number changes were identified using FACETS and validated by FISH. RNASeq was used to identify novel fusions genes and to identify differentially expressed genes. Genomic alterations associated with histological phenotype were identified. Genomic changes associated with recurrence and survival were identified by the Kaplan Meier method. Immortalized thyroid epithelial cell line, NTHY-ori 3.1 was used to express control and fusion gene for in vitro and in vivo experiments. Results: We elucidate the mutational profile and driver mutations in HCC and reveal that they exhibit a diverse spectrum of recurrent mutations, most of which have not been previously associated with this cancer (EIF1AX, MADCAM1). Notably, HCC harbor an extremely high prevalence of disruptive mutations to both protein-coding and tRNA encoding regions of the mitochondrial genome. We reveal unique chromosomal landscapes that involve whole chromosomal duplications of chromosomes 5 and 7 and wide spread major loss of heterozygosity arising from haploidization and copy number neutral uniparental disomy. These chromosomal processes underlie genetic instability and are highly prevalent in aggressive forms of HCC. We also identify novel fusion genes such as TMEM233/PRKAB1 which expression resulted in a transformation, organoids formation and invasion phenotype in vitro and tumorigenesis in vivo. These data suggested that TMEM233/PRKAB1 fusion plays as a critical driver and may serve as a therapeutic target for HCCs. Conclusions: We performed integrated genomic analysis of hurthle cell carcinoma revealing novel oncogenic drivers, recurrent mitochondrial mutations and unique chromosomal landscapes, which will help guide development of new treatments for one of the most deadly types of thyroid cancer. Citation Format: Yiyu Dong, Ian Ganly, Vladimir Makarov, Fengshen Kuo, Shyamprasad Deraje, Ed Reznik, Venkatraman Seshan, Gouri Nanjangud, Luc Morris, Nadeem Riaz, Eric Sherman, Ronald Ghossein, James Fagin, Timothy Chan. Integrated genomic analysis of Hurthle cell carcinoma reveals TMEM233/PRKAB1 fusion as a novel oncogenic driver [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 4619.

Chromosomal instability (CIN) is a hallmark of cancer and it results from ongoing errors in chromosome segregation during mitosis. While CIN is a major driver of tumor evolution, its role in metastasis has not been established. Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signaling. Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models, whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner. Using single-cell RNA sequencing, we uncover a CIN-induced transcriptional switch from a proliferative and metabolically active state to a mesenchymal phenotype associated with inflammatory pathways, offering an opportunity to target chromosome segregation errors for therapeutic benefit. Our work reveals an unexpected link between CIN, cytosolic DNA sensing pathways, and metastasis. The use of an isogenic system has enabled us to dissect the role of CIN from that of aneuploidy. Importantly, while we do not discount the role of CIN in generating karyotypic heterogeneity that can serve as the substrate for natural selection, our work demonstrates that continuous chromosome missegregation is also required to replenish cytosolic DNA pools leading to chronic upregulation of inflammatory pathways. In non-transformed settings, cytosolic DNA sensing is incompatible with viability. Unlike normal cells, chromosomally unstable cells are awash with cytosolic DNA and have adapted to coexist with a chronically active cGAS-STING pathway by suppressing downstream type I interferon signaling and instead upregulating the alternative NF-κB pathway. Persistent STING activation mediates carcinogen-induced tumor formation and we now show that tumor cells co-opt this otherwise lethal program to spread to distant organs. The evolutionary benefit of the noncanonical pathway might justify the scarcity of inactivating mutations in cGAS and STING among human cancers. The emergence, and subsequent tolerance, of CIN represents an important bottleneck during tumor evolution. Our single-cell analysis revealed that CIN induces a transcriptional switch whereby cells shift from a proliferative and highly metabolic state, ideally suited for primary tumor growth, to a chromosomally unstable and mesenchymal state associated with upregulation of inflammatory pathways. These two largely mutually exclusive states likely account for the reversibility in chromosome missegregation rates seen in primary tumors and metastases, and provide an explanation for the negative effect of aneuploidy during early tumorigenesis. Interestingly, this mutual exclusivity was recently observed in a pan-cancer genomic analysis of metastatic tumors, and it leads us to suggest that CIN underlies the subset of metastases that are characterized by EMT and inflammation. By providing a mechanistic link between CIN and metastasis, our work opens new avenues to target chromosomally unstable tumors for therapeutic benefit. Citation Format: Samuel F. Bakhoum, Bryon Ngo, Ashley L. Bakhoum, Julie-Ann Cavallo, Charles J. Murphy, Peter Ly, Pragya Shah, Roshan K. Sriram, Thomas B.k. Watkins, Neil K. Taunk, Mercedes Duran, Chantal Pauli, Christine Shaw, Kalyani Chadalavada, Vinagolu K. Rajasekhar, Giulio Genovese, Subramanian Venkatesan, Nicolai J. Birkbak, Nicholas McGranahan, Mark Lundquist, Quincy LaPlant, John H. Healey, Olivier Elemento, Christine H. Chung, Nancy Y. Lee, Marcin Imielinski, Gouri Nanjangud, Dana Pe'er, Don W. Cleveland, Simon N. Powell, Jan Lammerding, Charles Swanton, Lewis C. Cantley. Chromosomal instability promotes metastasis through a cytosolic DNA response [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 NG03.