Whole-genome landscape of pancreatic neuroendocrine tumours.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.

Whole-genome landscapes of major melanoma subtypes.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Corrigendum: Whole-genome landscape of pancreatic neuroendocrine tumours.

This corrects the article DOI: 10.1038/nature21063.

Genomic analyses identify molecular subtypes of pancreatic cancer.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Clinicopathologic and Genomic Characterization of PD-L1 Positive Urothelial Carcinomas.

INTRODUCTION: Pembrolizumab was approved with an accompanying companion diagnostic (CDx) assay (PD-L1 DAKO 22C3) for urothelial carcinoma (UC). In this study, we further characterize the clinicopathologic and genomic features of UC that are programmed death-ligand 1 (PD-L1) positive. MATERIALS AND METHODS: The cohort of this study consisted of a total of 528 consecutive UC patients with PD-L1 immunohistochemistry (IHC) and comprehensive genomic profiling (CGP). All PD-L1 IHC testing was performed using the DAKO 22C3 CDx assay for UC. PD-L1 positivity was determined at a combined positive score ≥ 10. RESULTS: A total of 44.5% (235/528) patients with UC were PD-L1positive . A lower PD-L1 positivity rate was detected in primary (42.3%, 148/350) versus metastatic sites (48.9%, 87/178). PD-L1 positivity was dependent on the location of the metastatic sites. CGP revealed PD-L1positive patients had more frequent genomic alterations (GAs) in TP53 (p = .006) and RB1 (p = .003) and less frequent GAs in FGFR3 (p = .001) and MTAP (p = .028). The APOBEC mutational signature and tumor mutational burden (TMB)-high were more common in PD-L1positive patients. By testing patients with UC with CGP, in addition to PD-L1 IHC, an additional 97 patients (18.4%) in the total cohort were eligible for immunotherapy based on TMB status. CONCLUSION: PD-L1positive and PD-L1negative urothelial carcinomas are genomically different. Also, our study provides the framework for future clinical investigation with regard to specimen site selection for PD-L1 testing as well as candidate biomarker genomic alterations that may predict for better response or lack of response to immune checkpoint inhibitors. IMPLICATIONS FOR PRACTICE: In this study, a higher prevalence of TP53 and RB1 alterations and APOBEC mutational signatures in the PD-L1positive urothelial carcinoma disease subset and enrichment of FGFR3 alterations in the PD-L1negative disease subset were found. These data provide the basis for future investigation into the role of these genomic changes as positive and negative predictors of immunotherapy response. Also, differences wer seen in PD-L1 positivity based on the collection site of the sample, which can provide a framework for future clinical trial design and could influence sample selection for PD-L1 testing in patients with urothelial carcinoma when multiple samples are available.

Primary neuroendocrine tumor of the pineal gland: a case report.

INTRODUCTION: Primary intracranial neuroendocrine tumors are exceedingly rare, with few cases in the literature. We present a case of a primary neuroendocrine carcinoma of the pineal gland, which is the second that has ever been reported. CASE PRESENTATION: A 53-year-old male patient presented with vomiting, weakness, and headaches. Imaging revealed a lesion in the pineal region, which was surgically resected. This mass was characterized by histology as a neuroendocrine carcinoma, given the presence of neuroendocrine markers and cytokeratin markers with absence of a primary lesion elsewhere on imaging. CONCLUSIONS: There are currently no guidelines on the management of primary intracranial neuroendocrine tumors. In this case, the patient underwent surgical resection and craniospinal radiotherapy. He subsequently received one cycle of chemotherapy with temozolomide, an alkylating agent, but he unfortunately did not tolerate treatment. A multidisciplinary decision was made along with the patient and his family to focus on palliative care. Eighteen months after the initial presentation, disease recurred in the patient's neck. The patient underwent resection to control the metastases, with a plan to follow with radiotherapy and chemotherapy. Unfortunately, the patient became unwell and died at 21 months after initial diagnosis. This demonstrates a need for continued research and reporting on this uncommon disease entity.

Whole genomes redefine the mutational landscape of pancreatic cancer.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Biomarkers in Breast Cancer: An Integrated Analysis of Comprehensive Genomic Profiling and PD-L1 Immunohistochemistry Biomarkers in 312 Patients with Breast Cancer.

BACKGROUND: We examined the current biomarker landscape in breast cancer when programmed death-ligand 1 (PD-L1) testing is integrated with comprehensive genomic profiling (CGP). MATERIAL AND METHODS: We analyzed data from samples of 312 consecutive patients with breast carcinoma tested with both CGP and PD-L1 (SP142) immunohistochemistry (IHC) during routine clinical care. These samples were stratified into hormone receptor positive (HR+)/human epidermal growth factor receptor negative (HER2-; n = 159), HER2-positive (n = 32), and triple-negative breast cancer (TNBC) cohorts (n = 121). RESULTS: We found that in the TNBC cohort, 43% (52/121) were immunocyte PD-L1-positive, and in the HR+/HER2- cohort, 30% (48/159) had PIK3CA companion diagnostics mutations, and hence were potentially eligible for atezolizumab plus nab-paclitaxel or alpelisib plus fulvestrant, respectively. Of the remaining 212 patients, 10.4% (22/212) had a BRCA1/2 mutation, which, if confirmed by germline testing, would allow olaparib plus talazoparib therapy. Of the remaining 190 patients, 169 (88.9%) were positive for another therapy-associated marker or a marker that would potentially qualify the patient for a clinical trial. In addition, we examined the relationship between immunocyte PD-L1 positivity and different tumor mutation burden (TMB) cutoffs and found that when a TMB cutoff of ≥9 mutations per Mb was applied (cutoff determined based on prior publication), 11.6% (14/121) patients were TMB ≥9 mutations/Mb and of these, TMB ≥9 mutations per Mb, 71.4% (10/14) were also positive for PD-L1 IHC. CONCLUSION: Our integrated PD-L1 and CGP methodology identified 32% of the tested patients as potentially eligible for at least one of the two new Food and Drug Administration approved therapies, atezolizumab or alpelisib, and an additional 61.2% (191/312) had other biomarker-guided potential therapeutic options. IMPLICATIONS FOR PRACTICE: This integrated programmed death-ligand 1 immunohistochemistry and comprehensive genomic profiling methodology identified 32% of the tested patients as eligible for at least one of the two new Food and Drug Administration-approved therapies, atezolizumab or alpelisib, and an additional 61.2% (191/312) had other biomarker-guided potential therapeutic options. These findings suggest new research opportunities to evaluate the predictive utility of other commonly seen PIK3CA mutations in hormone receptor-positive breast cancers and to standardize tumor mutation burden cutoffs to evaluate its potentially predictive role in triple-negative breast cancer.

Phenotypic and molecular dissection of metaplastic breast cancer and the prognostic implications.

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Telomere sequence content can be used to determine ALT activity in tumours.

The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.

Kava for generalised anxiety disorder: A 16-week double-blind, randomised, placebo-controlled study.

OBJECTIVE: Previous randomised, double-blind, placebo-controlled studies have shown that Kava (a South Pacific medicinal plant) reduced anxiety during short-term administration. The objective of this randomised, double-blind, placebo-controlled study was to perform a larger, longer-term trial assessing the efficacy and safety of Kava in the treatment of generalised anxiety disorder and to determine whether gamma-aminobutyric acid transporter (SLC6A1) single-nucleotide polymorphisms were moderators of response. METHODS: The trial was a phase III, multi-site, two-arm, 16-week, randomised, double-blind, placebo-controlled study investigating an aqueous extract of dried Kava root administered twice per day in tablet form (standardised to 120 mg of kavalactones twice/day) in 171 currently non-medicated anxious participants with diagnosed generalised anxiety disorder. The trial took place in Australia. RESULTS: An analysis of 171 participants revealed a non-significant difference in anxiety reduction between the Kava and placebo groups (a relative reduction favouring placebo of 1.37 points; p = 0.25). At the conclusion of the controlled phase, 17.4% of the Kava group were classified as remitted (Hamilton Anxiety Rating Scale score < 7) compared to 23.8% of the placebo group (p = 0.46). No SLC6A1 polymorphisms were associated with treatment response, while carriers of the rs2601126 T allele preferentially respond to placebo (p = 0.006). Kava was well tolerated aside from poorer memory (Kava = 36 vs placebo = 23; p = 0.044) and tremor/shakiness (Kava = 36 vs placebo = 23; p = 0.024) occurring more frequently in the Kava group. Liver function test abnormalities were significantly more frequent in the Kava group, although no participant met criteria for herb-induced hepatic injury. CONCLUSION: While research has generally supported Kava in non-clinical populations (potentially for more 'situational' anxiety as a short-term anxiolytic), this particular extract was not effective for diagnosed generalised anxiety disorder.

Whole genome sequencing of melanomas in adolescent and young adults reveals distinct mutation landscapes and the potential role of germline variants in disease susceptibility.

Cutaneous melanoma accounts for at least >10% of all cancers in adolescents and young adults (AYA, 15-30 years of age) in Western countries. To date, little is known about the correlations between germline variants and somatic mutations and mutation signatures in AYA melanoma patients that might explain why they have developed a cancer predominantly affecting those over 65 years of age. We performed genomic analysis of 50 AYA melanoma patients (onset 10-30 years, median 20); 25 underwent whole genome sequencing (WGS) of both tumor and germline DNA, exome data were retrieved from 12 TCGA AYA cases, and targeted DNA sequencing was conducted on 13 cases. The AYA cases were compared with WGS data from 121 adult cutaneous melanomas. Similar to mature adult cutaneous melanomas, AYA melanomas showed a high mutation burden and mutation signatures of ultraviolet radiation (UVR) damage. The frequencies of somatic mutations in BRAF (96%) and PTEN (36%) in the AYA WGS cohort were double the rates observed in adult melanomas (Q < 6.0 × 10-6 and 0.028, respectively). Furthermore, AYA melanomas contained a higher proportion of non-UVR-related mutation signatures than mature adult melanomas as a proportion of total mutation burden (p = 2.0 × 10-4 ). Interestingly, these non-UVR mutation signatures relate to APOBEC or mismatch repair pathways, and germline variants in related genes were observed in some of these cases. We conclude that AYA melanomas harbor some of the same molecular aberrations and mutagenic insults occurring in older adults, but in different proportions. Germline variants that may have conferred disease susceptibility correlated with somatic mutation signatures in a subset of AYA melanomas.

The stress response neuropeptide CRF increases amyloid-ß production by regulating γ-secretase activity.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-ß (Aß) production. In cells, CRF treatment increases Aß production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by ß-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aß. These data collectively link CRF to increased Aß through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aß and in some cases preferentially increase Aß42 via complex effects on γ-secretase.

Hypermutation In Pancreatic Cancer.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

Pediatric, Adolescent, and Young Adult Thyroid Carcinoma Harbors Frequent and Diverse Targetable Genomic Alterations, Including Kinase Fusions.

BACKGROUND: Thyroid carcinoma, which is rare in pediatric patients (age 0-18 years) but more common in adolescent and young adult (AYA) patients (age 15-39 years), carries the potential for morbidity and mortality. METHODS: Hybrid-capture-based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). RESULTS: GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety-three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. CONCLUSION: CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy. The Oncologist 2017;22:255-263 IMPLICATIONS FOR PRACTICE: The detection of diverse clinically relevant genomic alterations in the majority of pediatric, adolescent, and young adult patients with thyroid carcinoma in this study suggests that comprehensive genomic profiling may be beneficial for young patients with papillary, anaplastic, or medullary thyroid carcinoma, particularly for advanced or refractory cases for which clinical trials involving molecularly targeted therapies may be appropriate.

Activity of c-Met/ALK Inhibitor Crizotinib and Multi-Kinase VEGF Inhibitor Pazopanib in Metastatic Gastrointestinal Neuroectodermal Tumor Harboring EWSR1-CREB1 Fusion.

Malignant gastrointestinal neuroectodermal tumor (GNET) is an aggressive rare tumor, primarily occurring in young adults with frequent local-regional metastases and recurrence after local control. The tumor is characterized by the presence of EWSR1-ATF1 or EWSR1-CREB1 and immunohistochemical positivity for S-100 protein without melanocytic marker positivity. Due to poor responses to standard sarcoma regimens, GNET has a poor prognosis, and development of effective systemic therapy is desperately needed to treat these patients. Herein, we present a patient with a small bowel GNET who experienced recurrent hepatic and skeletal metastases after a primary resection. Comprehensive genomic profiling (CGP) in the course of clinical care with DNA and RNA sequencing demonstrated the presence of an exon 7 to exon 6 EWSR1-CREB1 fusion in the context of a diploid genome with no other genomic alterations. In a clinical trial, the patient received a combination of 250 mg crizotinib with 600 mg pazopanib quaque die and achieved partial response and durable clinical benefit for over 2.8 years, and with minimal toxicity from therapy. Using a CGP database of over 50,000 samples, we identified 11 additional cases that harbor EWSR1-CREB1 and report clinicopathologic characteristics, as these patients may also benefit from such a regimen.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.

Pen-2 is essential for γ-secretase complex stability and trafficking but partially dispensable for endoproteolysis.

The 19-transmembrane γ-secretase complex generates the amyloid ß-peptide of Alzheimer's disease by intramembrane proteolysis of the ß-amyloid precursor protein. This complex is comprised of presenilin, Aph1, nicastrin, and Pen-2. The exact function and mechanism of the highly conserved Pen-2 subunit remain poorly understood. Using systematic mutagenesis, we confirm and extend our understanding of which key regions and specific residues play roles in various aspects of γ-secretase function, including maturation, localization, and activity, but not processivity. In general, mutations (1) within the first half of transmembrane domain (TMD) 1 of Pen-2 decreased PS1 endoproteolysis and γ-secretase proteolytic activity, (2) within the second half of TMD1 increased proteolytic activity, (3) within the cytosolic loop region decreased proteolytic activity, (4) within TMD2 decreased PS1 endoproteolysis, (5) within the first half of TMD2 decreased proteolytic activity, and (6) within C-terminal residues decreased proteolytic activity. Specific mutational effects included N33A in TMD1 causing an increase in γ-secretase complexes at the cell surface and a modest decrease in stability and the previously unreported I53A mutation in the loop region reducing stability 10-fold and proteolytic activity by half. In addition, we confirm that minor PS1 endoproteolysis can occur in the complete absence of Pen-2. Together, these data suggest that rather than solely being a catalyst for γ-secretase endoproteolysis, Pen-2 may also stabilize the complex prior to PS1 endoproteolysis, allowing time for full assembly and proper trafficking.

Functional analysis and purification of a Pen-2 fusion protein for γ-secretase structural studies.

The 19-transmembrane, multisubunit γ-secretase complex generates the amyloid ß-peptide (Aß) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the ß-amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal maltose binding protein tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex.