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.

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.

Precision Oncology in Surgery: Patient Selection for Operable Pancreatic Cancer.

OBJECTIVE: We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection. BACKGROUND: Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease. METHOD: In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram. RESULTS: High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables. CONCLUSIONS: Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.

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.

Genomic analysis of HPV-positive versus HPV-negative oesophageal adenocarcinoma identifies a differential mutational landscape.

BACKGROUND: High-risk human papillomavirus (hr-HPV) has been implicated in a subset of patients with oesophageal adenocarcinoma (OAC). We therefore hypothesised that HPV associated OAC may have distinct genomic aberrations compared with viral negative oesophageal cancer. METHODS: Whole exome sequencing was performed to explore the mutational landscape and potential molecular signature of HPV-positive versus HPV-negative OAC. Four hr-HPV-positive and 8 HPV-negative treatment-naive fresh-frozen OAC tissue specimens and matched normal tissue were analysed to identify somatic genomic mutations. Data were subjected to cancer driver gene identification and pathway analysis. RESULTS: The HPV-positive cohort harboured approximately 50% less non-silent somatic mutations than the virus-negative patients with oesophageal cancer (1.31 mutations/Mb vs 2.56 mutations/Mb, p=0.048). TP53 aberrations were absent in the HPV-positive OAC group whereas 50% of the HPV-negative patients with OAC exhibited TP53 mutations. HPV-negative cancers were enriched with non-silent mutations in cancer driver genes, but not HPV-positive tumours. Enriched A>C transversions at adenine-adenine (AA) dinucleotide was observed in 5/7 Siewert class I OAC samples but none (0/5) in Siewert class II tumours (p=0.027). CONCLUSIONS: These findings demonstrate distinct genomic differences between HPV-positive and HPV-negative OACs indicating different biological mechanisms of tumour formation.

Diagnosis and Management of Hereditary Pancreatic Cancer.

Hereditary pancreatic cancer can be diagnosed through family history and/or a personal history of pancreatitis or clinical features suggesting one of the known pancreatic cancer predisposition syndromes. This chapter describes the currently known hereditary pancreatic cancer predisposition syndromes, including Peutz-Jeghers syndrome, familial atypical multiple mole melanoma, hereditary breast and ovarian cancer, Li-Fraumeni syndrome, hereditary non-polyposis colon cancer and familial adenomatous polyposis. Strategies for genetic testing for hereditary pancreatic cancer and the appropriate options for surveillance and cancer risk reduction are discussed. Finally, ongoing research and future directions in the diagnosis and management of hereditary pancreatic cancer will be considered.

Clinical and pathologic features of familial pancreatic cancer.

BACKGROUND: Inherited predisposition to pancreatic cancer contributes significantly to its incidence and presents an opportunity for the development of early detection strategies. The genetic basis of predisposition remains unexplained in a high proportion of patients with familial PC (FPC). METHODS: Clinicopathologic features were assessed in a cohort of 766 patients who had been diagnosed with pancreatic ductal adenocarcinoma (PC). Patients were classified with FPC if they had ≥1 affected first-degree relatives; otherwise, they were classified with sporadic PC (SPC). RESULTS: The prevalence of FPC in this cohort was 8.9%. In FPC families with an affected parent-child pair, 71% in the subsequent generation were 12.3 years younger at diagnosis. Patients with FPC had more first-degree relatives who had an extrapancreatic malignancy (EPM) (42.6% vs 21.2; P<.0001), particularly melanoma and endometrial cancer, but not a personal history of EPM. Patients with SPC were more likely to be active smokers, have higher cumulative tobacco exposure, and have fewer multifocal precursor lesions, but these were not associated with differences in survival. Long-standing diabetes mellitus (>2 years) was associated with poor survival in both groups. CONCLUSIONS: FPC represents 9% of PC, and the risk of malignancy in kindred does not appear to be confined to the pancreas. Patients with FPC have more precursor lesions and include fewer active smokers, but other clinicopathologic factors and outcome are similar to those in patients with SPC. Furthermore, some FPC kindreds may exhibit anticipation. A better understanding of the clinical features of PC will facilitate efforts to uncover novel susceptibility genes and the development of early detection strategies.

Drug-induced liver injury from selective androgen receptor modulators, anabolic-androgenic steroids and bodybuilding supplements in Australia.

BACKGROUND: Reports of DILI due to herbal and dietary supplements have been increasing over time. AIMS: To characterise clinical, laboratory and histopathological phenotypes and outcomes of drug-induced liver injury (DILI) due to anabolic-androgenic steroids (AAS), selective androgen receptor modulators (SARMs), and bodybuilding supplements (BBS) in Australia. METHODS: Retrospective case series. Patients presented to nine Australian tertiary hospitals, 2017-2023. DILI was defined biochemically and patients were included if their treating physician attributed DILI to preceding use of AAS, SARMs or BBS. Primary endpoint was time to normalisation of liver biochemistry. Secondary endpoints were hospitalisation for investigation or management of DILI, death attributable to liver injury, and liver transplantation. RESULTS: Twenty-three cases of DILI were identified, involving 40 drugs: 18 AAS, 14 SARMs and eight BBS. Patients were predominantly male (22/23), with median age 30 years (IQR 26-42). Most were symptomatic (21/23). Median latency of onset was 58 days (IQR 28-112 days) from drug commencement. Most patients (17/23) were admitted to hospital. Based on updated Roussel Uclaf Causality Assessment Method, DILI was possible in 17/23, probable in 2/23 and unlikely in 4/23. Median time to normalisation of liver biochemistry was 175 days (IQR 70-292 days) from presentation. Three (3/23) were treated with corticosteroids, 14/23 were treated for itch, and one (1/23) underwent liver transplantation. There were no deaths. CONCLUSIONS: The prognosis of DILI from AAS, SARMs and BBS is good although liver transplantation may rarely be required. A detailed drug history is important in uncovering DILI due to these supplements.

Status of confocal laser endomicroscopy in gastrointestinal disease.

Confocal laser endomicroscopy (CLE) is an advanced imaging technique which combines conventional white light endoscopy (WLE) with an integrated or probe based confocal microscope. This allows microscopic examination of the surface epithelium and in vivo diagnosis during endoscopy. Established CLE applications include the diagnosis of Barrett's oesophagus, gastric intestinal metaplasia, coeliac disease and microscopic colitis. CLE can differentiate hyperplastic from adenomatous polyps in the colon and may obviate the need to biopsy all polyps at endoscopy. CLE is particularly helpful in surveillance endoscopy in inflammatory bowel disease where it has been shown to reduce the number of biopsies required and improve the detection of dysplasia. The future of CLE may be with new contrast agents to allow for molecular tagging and improved endoscopic diagnoses. The aim of this review is to describe the technology and techniques involved in CLE, and discuss the evolving applications in obtaining "virtual biopsy" throughout the GI tract.

Subepithelial mass lesions in the upper gastrointestinal tract.

The finding of a mass lesion in the upper gastrointestinal tract at endoscopy with apparent normal overlying mucosa is common. The differential diagnosis of such lesions is broad and includes those of intramural or extramural origin. Endoscopic ultrasound provides accurate imaging of subepithelial mass lesions and characterizes them according to size, echogenicity, and origin including the histologic layer if the lesion is intramural which narrows the differential diagnosis. Endoscopic ultrasound allows a guided tissue sample to be obtained for histologic confirmation which is especially important for hypoechoic lesions arising from the 3rd or 4th echogenic layers. The purpose of this article is to review the diagnosis of the more common subepithelial mass lesions with an emphasis on endoscopic ultrasound and the subsequent management or monitoring.

Lost in translation: returning germline genetic results in genome-scale cancer research.

BACKGROUND: The return of research results (RoR) remains a complex and well-debated issue. Despite the debate, actual data related to the experience of giving individual results back, and the impact these results may have on clinical care and health outcomes, is sorely lacking. Through the work of the Australian Pancreatic Cancer Genome Initiative (APGI) we: (1) delineate the pathway back to the patient where actionable research data were identified; and (2) report the clinical utilisation of individual results returned. Using this experience, we discuss barriers and opportunities associated with a comprehensive process of RoR in large-scale genomic research that may be useful for others developing their own policies. METHODS: We performed whole-genome (n = 184) and exome (n = 208) sequencing of matched tumour-normal DNA pairs from 392 patients with sporadic pancreatic cancer (PC) as part of the APGI. We identified pathogenic germline mutations in candidate genes (n = 130) with established predisposition to PC or medium-high penetrance genes with well-defined cancer associated syndromes or phenotypes. Variants from candidate genes were annotated and classified according to international guidelines. Variants were considered actionable if clinical utility was established, with regard to prevention, diagnosis, prognostication and/or therapy. RESULTS: A total of 48,904 germline variants were identified, with 2356 unique variants undergoing annotation and in silico classification. Twenty cases were deemed actionable and were returned via previously described RoR framework, representing an actionable finding rate of 5.1%. Overall, 1.78% of our cohort experienced clinical benefit from RoR. CONCLUSION: Returning research results within the context of large-scale genomics research is a labour-intensive, highly variable, complex operation. Results that warrant action are not infrequent, but the prevalence of those who experience a clinical difference as a result of returning individual results is currently low.

Precision Medicine for Advanced Pancreas Cancer: The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) Trial.

PURPOSE: Personalized medicine strategies using genomic profiling are particularly pertinent for pancreas cancer. The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial was initially designed to exploit results from genome sequencing of pancreatic cancer under the auspices of the International Cancer Genome Consortium (ICGC) in Australia. Sequencing revealed small subsets of patients with aberrations in their tumor genome that could be targeted with currently available therapies. EXPERIMENTAL DESIGN: The pilot stage of the IMPaCT trial assessed the feasibility of acquiring suitable tumor specimens for molecular analysis and returning high-quality actionable genomic data within a clinically acceptable timeframe. We screened for three molecular targets: HER2 amplification; KRAS wild-type; and mutations in DNA damage repair pathways (BRCA1, BRCA2, PALB2, ATM). RESULTS: Tumor biopsy and archived tumor samples were collected from 93 patients and 76 were screened. To date 22 candidate cases have been identified: 14 KRAS wild-type, 5 cases of HER2 amplification, 2 mutations in BRCA2, and 1 ATM mutation. Median time from consent to the return of validated results was 21.5 days. An inability to obtain a biopsy or insufficient tumor content in the available specimen were common reasons for patient exclusion from molecular analysis while deteriorating performance status prohibited a number of patients from proceeding in the study. CONCLUSIONS: Documenting the feasibility of acquiring and screening biospecimens for actionable molecular targets in real time will aid other groups embarking on similar trials. Key elements include the need to better prescreen patients, screen more patients, and offer more attractive clinical trial options.

Returning individual research results for genome sequences of pancreatic cancer.

BACKGROUND: Disclosure of individual results to participants in genomic research is a complex and contentious issue. There are many existing commentaries and opinion pieces on the topic, but little empirical data concerning actual cases describing how individual results have been returned. Thus, the real life risks and benefits of disclosing individual research results to participants are rarely if ever presented as part of this debate. METHODS: The Australian Pancreatic Cancer Genome Initiative (APGI) is an Australian contribution to the International Cancer Genome Consortium (ICGC), that involves prospective sequencing of tumor and normal genomes of study participants with pancreatic cancer in Australia. We present three examples that illustrate different facets of how research results may arise, and how they may be returned to individuals within an ethically defensible and clinically practical framework. This framework includes the necessary elements identified by others including consent, determination of the significance of results and which to return, delineation of the responsibility for communication and the clinical pathway for managing the consequences of returning results. RESULTS: Of 285 recruited patients, we returned results to a total of 25 with no adverse events to date. These included four that were classified as medically actionable, nine as clinically significant and eight that were returned at the request of the treating clinician. Case studies presented depict instances where research results impacted on cancer susceptibility, current treatment and diagnosis, and illustrate key practical challenges of developing an effective framework. CONCLUSIONS: We suggest that return of individual results is both feasible and ethically defensible but only within the context of a robust framework that involves a close relationship between researchers and clinicians.

Clinical and molecular characterization of HER2 amplified-pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies. METHODS: HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC). RESULTS: An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum. CONCLUSIONS: HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.

Histomolecular phenotypes and outcome in adenocarcinoma of the ampulla of vater.

PURPOSE: Individuals with adenocarcinoma of the ampulla of Vater demonstrate a broad range of outcomes, presumably because these cancers may arise from any one of the three epithelia that converge at that location. This variability poses challenges for clinical decision making and the development of novel therapeutic strategies. PATIENTS AND METHODS: We assessed the potential clinical utility of histomolecular phenotypes defined using a combination of histopathology and protein expression (CDX2 and MUC1) in 208 patients from three independent cohorts who underwent surgical resection for adenocarcinoma of the ampulla of Vater. RESULTS: Histologic subtype and CDX2 and MUC1 expression were significant prognostic variables. Patients with a histomolecular pancreaticobiliary phenotype (CDX2 negative, MUC1 positive) segregated into a poor prognostic group in the training (hazard ratio [HR], 3.34; 95% CI, 1.69 to 6.62; P < .001) and both validation cohorts (HR, 5.65; 95% CI, 2.77 to 11.5; P < .001 and HR, 2.78; 95% CI, 1.25 to 7.17; P = .0119) compared with histomolecular nonpancreaticobiliary carcinomas. Further stratification by lymph node (LN) status defined three clinically relevant subgroups: one, patients with histomolecular nonpancreaticobiliary (intestinal) carcinoma without LN metastases who had an excellent prognosis; two, those with histomolecular pancreaticobiliary carcinoma with LN metastases who had a poor outcome; and three, the remainder of patients (nonpancreaticobiliary, LN positive or pancreaticobiliary, LN negative) who had an intermediate outcome. CONCLUSION: Histopathologic and molecular criteria combine to define clinically relevant histomolecular phenotypes of adenocarcinoma of the ampulla of Vater and potentially represent distinct diseases with significant implications for current therapeutic strategies, the ability to interpret past clinical trials, and future trial design.