Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

A predictive model of inflammatory markers and patient-reported symptoms for cachexia in newly diagnosed pancreatic cancer patients.

BACKGROUND: Cachexia is a frequent manifestation of pancreatic cancer, can limit a patient's ability to take chemotherapy, and is associated with shortened survival. We developed a model to predict the early onset of cachexia in advanced pancreatic cancer patients. METHODS: Patients with newly diagnosed, untreated metastatic or locally advanced pancreatic cancer were included. Serum cytokines were drawn prior to therapy. Patient symptoms were recorded using the M.D. Anderson Symptom Inventory (MDASI). Our primary endpoint was either 10% weight loss or death within 60 days of the start of therapy. RESULTS: Twenty-seven of 89 patients met the primary endpoint (either having lost 10% of body weight or having died within 60 days of the start of treatment). In a univariate analysis, smoking, history symptoms of pain and difficulty swallowing, high levels of MK, CXCL-16, IL-6, TNF-a, and low IL-1b all correlated with this endpoint. We used recursive partition to fit a regression tree model, selecting four of 26 variables (CXCL-16, IL-1b, pain, swallowing difficulty) as important in predicting cachexia. From these, a model of two cytokines (CXCL-16 > 5.135 ng/ml and IL-1b < 0.08 ng/ml) demonstrated a better sensitivity and specificity for this outcome (0.70 and 0.86, respectively) than any individual cytokine or tumor marker. CONCLUSIONS: Cachexia is frequent in pancreatic cancer; one in three patients met our endpoint of 10% weight loss or death within 60 days. Inflammatory cytokines are better than conventional tumor markers at predicting this outcome. Recursive partitioning analysis suggests that a model of CXCL-16 and IL-1B may offer a better ability than individual cytokines to predict this outcome.

Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes.

BACKGROUND: The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies. PATIENTS AND METHODS: We isolated shed exosomes in biofluids from three patients with pancreaticobiliary cancers (two pancreatic, one ampullary). We performed comprehensive profiling of exoDNA and exoRNA by whole genome, exome and transcriptome sequencing using the Illumina HiSeq 2500 sequencer. We assessed the feasibility of calling copy number events, detecting mutational signatures and identifying potentially actionable mutations in exoDNA sequencing data, as well as expressed point mutations and gene fusions in exoRNA sequencing data. RESULTS: Whole-exome sequencing resulted in 95%-99% of the target regions covered at a mean depth of 133-490×. Genome-wide copy number profiles, and high estimates of tumor fractions (ranging from 56% to 82%), suggest robust representation of the tumor DNA within the shed exosomal compartment. Multiple actionable mutations, including alterations in NOTCH1 and BRCA2, were found in patient exoDNA samples. Further, RNA sequencing of shed exosomes identified the presence of expressed fusion genes, representing an avenue for elucidation of tumor neoantigens. CONCLUSIONS: We have demonstrated high-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers. A wide range of cancer-derived biomarkers could be detected within the nucleic acid cargo of shed exosomes, including copy number profiles, point mutations, insertions, deletions, gene fusions and mutational signatures. Liquid biopsies using shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.

Validation of histomolecular classification utilizing histological subtype, MUC1, and CDX2 for prognostication of resected ampullary adenocarcinoma.

BACKGROUND: Outcomes for ampullary adenocarcinomas are heterogeneous, and numerous methods of categorisation exist. A histomolecular phenotype based on histology, caudal-type homeodomain transcription factor 2 (CDX2) staining and Mucin 1 (MUC1) staining has recently been tested and validated in two cohorts. We attempt to validate this classification in a large patient population. METHODS: Tissue samples from 163 patients with resected ampullary adenocarcinoma were classified based on histology and immunohistochemical expression of CDX2 and MUC1. A pancreaticobiliary histomolecular classification (PB) was defined as a sample with pancreaticobiliary histology, positive MUC1 and negative CDX2 expression. RESULTS: There were 82 deaths; median follow-up of 32.4 months; and median overall survival of 87.7 (95% CI 42.9-109.5) months. PB comprised 28.2% of the cases. Factors associated with overall survival were histological subtype (P=0.0340); T1/2 vs T3/4 (P=0.001); perineural (P<0.0001) and lymphovascular (P=0.0203) invasion; and histomolecular intestinal histomolecular phenotype (INT) vs PB phenotype (106.4 vs 21.2 months, P<0.0001). Neither MUC1 nor CDX2 was statistically significant, although MUC1 positivity defined as ⩾10% staining was significant (P=0.0023). In multivariate analysis, age (HR 1.03), PB phenotype (HR 2.26) and perineural invasion (PNI; HR 2.26) were associated with poor survival. CONCLUSIONS: The prognostic ability of histomolecular phenotype has been validated in an independent cohort of ampullary adenocarcinoma patients.

Carcinoma of unknown primary with gastrointestinal profile: immunohistochemistry and survival data for this favorable subset.

BACKGROUND: Carcinoma of unknown primary with a "gastrointestinal profile" is an emerging, favorable entity. Distinguishing this entity is of increasing significance given the progress in the treatment of colorectal cancer. PATIENTS AND METHODS: 74 carcinoma of unknown primary (CUP) patients with CDX2+ tumors were chosen from the databases at M.D. Anderson and Sarah Cannon Cancer Centers between 2004 and 2010. Data on clinical and pathological characteristics including therapy and survival were recorded. RESULTS: 20 patients had ascites on presentation; the predominant sites of metastases included liver (30 %), carcinomatosis (50 %), and nodes (51 %). Based on immunohistochemistry, 2 cohorts were created: Cohort 1-"consistent with lower GI profile" included 34 patients [CDX-2+, CK20+, CK7-] and Cohort 2-"probable lower GI profile" included 40 patients [CDX2+, irrespective of CK7/CK20 status]. Excluding 6 outliers, Cohorts 1 and 2 had 32 and 36 patients, respectively; their median survivals were 37 and 21 months, respectively. On multivariate Cox regression analysis, only liver metastases were found to negatively influence survival. CONCLUSIONS: Our retrospective study provides encouraging indications that CUP patients with gastrointestinal profiles benefit from site-specific therapy. We recommend all CUP patients, especially those with abdominal nodes, isolated carcinomatosis or liver metastases, to undergo optimal immunohistochemistry (IHC) to check for a gastrointestinal profile of CUP.

A retrospective study of ampullary adenocarcinomas: overall survival and responsiveness to fluoropyrimidine-based chemotherapy.

BACKGROUND: Whether carcinomas of the ampulla of Vater should be classified with biliary tract tumors and treated in a similar manner remains unknown. We sought to compare the outcomes of similarly staged periampullary adenocarcinomas (AAs) and analyze the chemotherapy responsiveness of AAs. PATIENTS AND METHODS: A total of 905 patients with resected periampullary adenocarcinomas were identified from a prospective surgical registry from 1988 to 2010. A second cohort of 64 metastatic AA patients from 1992 to 2009 who received either front-line fluoropyrimidine-based or gemcitabine-based chemotherapy was also identified. RESULTS: Overall survival (OS) for AAs was similar to survival with duodenal adenocarcinomas, but was significantly different from both extrahepatic biliary and pancreatic adenocarcinomas (P < 0.001 for each comparison). In multivariate analysis, AAs had a significantly improved OS in comparison with extrahepatic biliary adenocarcinomas (HR = 1.97, P = 0.006). Fluoropyrimidine-based as opposed to gemcitabine-based chemotherapy for metastatic AAs resulted in a significant improvement in time to progression (P = 0.001) but only a trend toward benefit for OS (P = 0.07) in multivariate analysis. CONCLUSIONS: Differences in the natural history of ampullary and extrahepatic biliary adenocarcinomas exist. Analyses of metastatic ampullary adenocarcinomas suggest that fluoropyrimidine-based chemotherapy may represent a more appropriate front-line chemotherapy approach.

Carcinoid tumours: predicting the location of the primary neoplasm based on the sites of metastases.

OBJECTIVES: To predict the primary neuroendocrine tumour of the gastrointestinal tract site based on observed metastatic sites. METHODS: We studied data from the radiology database of a single, large cancer centre on 250 patients with pathologically confirmed neuroendocrine tumours. Primary tumour sites and the locations of metastases were collected from pathologic and radiologic reports of all available imaging modalities, such as computed tomography (CT), positron emission tomography (PET/CT), magnetic resonance imaging (MRI) and octreotide scans in the database. A nominal regression model was used to predict primary tumour site using the observed metastatic sites. Regression coefficients that were not statistically significant at the 5 % level were eliminated from the model in a stepwise procedure. RESULTS: Lung and liver metastases were not statistically significant predictors of the location of primary tumours (p = 0.86 and 0.074, respectively); whereas, lymph node, bone, and peritoneal metastases were significant predictors (p < 0.0001, 0.0004, and 0.014, respectively). CONCLUSIONS: Metastatic neuroendocrine tumours to the lymph nodes, bone, and peritoneum can be used to predict the primary neuroendocrine site; however, metastases in the lung and liver alone cannot predict the site of the primary tumour site.

CT imaging features of acinar cell carcinoma and its hepatic metastases.

OBJECTIVE: To evaluate and describe the computed tomography features of pure acinar cell carcinoma (ACC) and its liver metastases. METHODS: Thirty patients were evaluated. Two radiologists evaluated imaging findings for each tumor for size, location, internal density, enhancement, tumor calcifications, pancreatic, and common biliary ductal obstructions and metastases. RESULTS: 70 % were male. Fourteen tumors were located in the pancreatic head, 14 in the tail, one in the neck, and one in the uncinate process. Abdominal pain was the most common presenting symptom (93 %), 20 % had pancreatitis and 17 % had obstructive jaundice. The average tumor size was 7 cm, 97 % of tumors were solid, well circumscribed (73 %); isodense to normal pancreatic parenchyma (40 %) on the non-contrast study, hypodense on the arterial (47 %), and hypodense on the portal venous (37 %) phase. 30 % patients had pancreatic ductal dilation, 10 % had pancreatic ductal ingrowth, 6 % had calcifications, and 20 % had central necrosis, and 31 % (5/16) showed biliary ductal dilation. At presentation, 50 % had metastatic adenopathy and 40 % patients had liver metastases, which typically were well circumscribed, hypoattenuating to the hepatic parenchyma on all the phases of contrast enhancement and had a lobulated margin. CONCLUSION: ACCs of the pancreas often present as large, well circumscribed, solid masses commonly in males. Despite their large size, they may not cause CBD obstruction.

Cancer of unknown primary: progress in the search for improved and rapid diagnosis leading toward superior patient outcomes.

This paper explores the enigma of cancer of unknown primary (CUP) in relation to rapidly improving molecular diagnostic approaches. It is based on the first global collaboration meeting on improving research and clinical outcomes in CUP organized by the CUP Foundation. We review the difficulties of classifying this widely heterogeneous disease and the available diagnostic and pathological evaluative techniques, focusing on molecular profiling. Retrospective studies in CUP patients are shown to provide indirect validation of the accuracy of several platforms of gene expression profiling assays that may identify CUP subsets that respond favorably to active chemotherapy regimens. This review concludes that the recent major improvements in pathologic and molecular diagnostics, coupled with new improved therapies for several specific advanced solid tumors, need to be harmonized with more evidence from clinical-translational trials. All patients with CUP could thus be appropriately managed without the constant uncertainty that has previously severely hampered patient care and optimal outcomes. The longer-term objective is to understand the biology of highly metastatic disease, leading to the development of future global therapeutic programs. Current clinical studies, such as CUP-ONE, will address some of these issues.

The integration of chemoradiation in the care of patient with localized pancreatic cancer.

The use of chemoradiation for patients with localized pancreatic cancer is controversial. Although some randomized trials have indicated that chemoradiation improves the median survival of patients with locally advanced as well as resected pancreatic cancer, other more recent trials have called into question the role of chemoradiation and have supported the use of chemotherapy. In the adjuvant setting, the high local tumor recurrence/persistence rate in all trials probably reflects the inclusion of patients with incompletely resected tumors, whose prognosis is similar to the prognosis of patients with locally advanced who do not undergo resection, making these trials difficult to interpret. More precise clinical staging and selection of patients appropriate for surgical resection is an important goal. The keys to the successful integration of radiotherapy in the care of patients with localized pancreatic cancer are selection, sequencing and smaller treatment volumes. A strategy of initial chemotherapy followed by consolidation with a well-tolerated chemoradiation regimen both in the adjuvant and locally advanced settings maximizes benefits of both treatment options, which are in fact complementary. Herein, we discuss the rationale for this approach as well as the ongoing investigation of novel radiation approaches designed to enhance outcome through the molecular and physical targeting of disease as well as the investigation of neoadjuvant chemoradiation in radiographically resectable and borderline resectable pancreatic cancer.

Spontaneous pneumothorax in malignancy: a case report and review of the literature.

BACKGROUND: Pneumothorax occurring in the absence of obvious lung disease is defined as spontaneous pneumothorax. Spontaneous pneumothorax occurs in a variety of settings in patients with malignancies. PATIENTS AND METHODS: We present a case report of spontaneous pneumothorax in malignancy and review the literature. RESULTS: No correlation was found between the occurrence of pneumothorax with age, sex or smoking history. Pneumothorax occurred with a variety of primary tumors. However it was always associated with lung metastases or lung involvement with tumor. In certain cases the metastases were detected after the occurrence of pneumothorax. CONCLUSIONS: The occurrence of pneumothorax in a patient with malignancy should prompt a search for lung metastases.