Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed. DESIGN: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites. RESULTS: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.

A fully 3D-printed versatile tumor-on-a-chip allows multi-drug screening and correlation with clinical outcomes for personalized medicine.

Optimal clinical outcomes in cancer treatments could be achieved through the development of reliable, precise ex vivo tumor models that function as drug screening platforms for patient-targeted therapies. Microfluidic tumor-on-chip technology is emerging as a preferred tool since it enables the complex set-ups and recapitulation of the physiologically relevant physical microenvironment of tumors. In order to overcome the common hindrances encountered while using this technology, a fully 3D-printed device was developed that sustains patient-derived multicellular spheroids long enough to conduct multiple drug screening tests. This tool is both cost effective and possesses four necessary characteristics of effective microfluidic devices: transparency, biocompatibility, versatility, and sample accessibility. Compelling correlations which demonstrate a clinical proof of concept were found after testing and comparing different chemotherapies on tumor spheroids, derived from ten patients, to their clinical outcomes. This platform offers a potential solution for personalized medicine by functioning as a predictive drug-performance tool.

A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.

Introduction: Ex vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy. Methods: A multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 µM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients' clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST). Results: The cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4-0.97) and a sensitivity of 96% (24/25, 95% CI 0.80-0.99). Conclusion: EVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.

A universal cell-free DNA approach for response prediction to preoperative chemoradiation in rectal cancer.

The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.

Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics.

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

Methionine aminopeptidase 2 as a potential target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive metastatic cancer with a very low survival rate. This tumor is hypovascularized and characterized by severe hypoxic regions, yet these regions are not impeded by the oxidative stress in their microenvironment. PDA's high resilience raises the need to find new effective therapeutic targets. This study investigated the suitability of methionine aminopeptidase 2 (MetAp2), a metallopeptidase known to play an important role in tumor progression, as a new target for treating PDA. In our examination of patient-derived PDA tissues, we found that MetAp2 is highly expressed in metastatic regions compared with primary sites. At the cellular level, we found that the basal expression levels of MetAp2 in pancreatic cancer cells were higher than its levels in endothelial cells. Pancreatic cancer cells showed a significant suppression of proliferation in a dose-dependent manner upon exposure to TNP-470, a selective MetAp2 inhibitor. In addition, a significant reduction in glutathione (GSH) levels - known for its importance in alleviating oxidative stress - was detected in all treated cells, suggesting a possible anti-cancer activity mechanism that would be feasible for treating highly hypoxic PDA tumors. Furthermore, in an orthotopic pancreatic cancer murine model, systemic oral treatment with a MetAp2 inhibitor significantly reduced tumors' growth. Taken together, our findings indicate that MetAp2 enhances tumor sensitivity to hypoxia and may provide an effective target for treating hypoxic tumors with high expression levels of MetAp2.

Liquid biopsy reveals collateral tissue damage in cancer.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

[THE MAGIC OF IMMUNOTHERAPY - ANTI-PD1 TREATMENT IN ADENOCARCINOMA OF THE GASTROESOPHAGEAL JUNCTION].

INTRODUCTION: Adenocarcinomas of the gastroesophageal junction (GEJ) are tumors whose incidence rates increased significantly in recent years. These tumors have a poor prognosis, and are treated with a palliative approach when diagnosed in an advanced stage. Some tumors overexpress PDL-1 proteins which are partially responsible for the "immune escape" of tumor cells. However, this over-expression enables the use of an immunotherapeutic treatment approach. Immunotherapy using anti-PD1/PDL-1 shows promising results in patients with metastatic GEJ tumors. In the present case report, we describe a young patient who was diagnosed with a non-resectable GEJ adenocarcinoma and was found to have almost 100% positivity for PDL-1 in the tumor biopsy, using standard immunohistochemistry. The patient had rapid and complete response to the anti-PD1 antibody treatment with further development of immune mediated side effects.

ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

Clinical Characteristics and Prognosis of Gastric Cancer Patients with BRCA 1/2 Germline Mutations: Report of Ten Cases and a Literature Review.

BACKGROUND: The prognosis of gastric cancer (GC) is poor with a median overall survival (OS) of less than 12 months in advanced-stage disease. The search for distinct genetic subgroups of GC patients and predictive biomarkers is ongoing. While BRCA1 or BRCA2 germline mutations (gBRCAm) have potential therapeutic implications in ovarian, breast and pancreatic cancers, their significance in GC patients has not been established. PATIENTS AND METHODS: A retrospective multi-center data analysis of GC patients with gBRCAm was conducted, detailing the clinical characteristics and disease course in this unique subset of patients. RESULTS: Ten GC patients with gBRCAm were identified, six of them with metastatic disease. The median OS of all ten GC patients was 47.5 (13-192) months. Median OS for patients diagnosed with operable disease was 55.5 (13-192) months and of the patients with metastatic disease (calculated from metastatic disease diagnosis) 32 (15-52) months with an exceptional 1-, 2- and 3-year survival rate of 100%, 83.3% and 50%, respectively. CONCLUSION: These preliminary data suggest that gBRCAm in GC patients are associated with a favorable prognosis. Furthermore, gBRCAm might be a predictive biomarker to DNA-damaging agents response in GC patients, similarly to its established role in other malignancies. Further research is needed to confirm our findings.

Rapid Clearing for High Resolution 3D Imaging of Ex Vivo Pancreatic Cancer Spheroids.

The currently accepted imaging methods have been a central hurdle to imaging the finer details of tumor behavior in three-dimensional (3D) ex vivo multicellular culture models. In our search for an improved way of imaging tumor behavior in its physiological-like niche, we developed a simple, efficient, and straightforward procedure using standard reagents and imaging equipment that significantly enhanced 3D imaging up to a ~200-micron depth. We tested its efficacy on pancreatic spheroids, prototypes of high-density tissues that are difficult to image. We found we could both save time with this method and extract information about pancreatic tumor spheroids that previously was difficult to obtain. We were able to discern clear differences in the organization of pancreatic tumor spheroids generated from different origins, suggesting cell-specific, inherent, bottom-up organization with a correlation to the level of malignancy. We also examined the dynamic changes in the spheroids at predetermined time points, providing important information related to tissue morphogenesis and its metabolic state. Lastly, this process enabled us to assess a drug vehicle's potential to penetrate dense tumor tissue by improving our view of the inert particles' diffusion in the 3D spheroid. This clearing method, a simple procedure, can open the door to more accurate imaging and reveal more about cancer behavior.

Corrigendum to "Mucinous Histology, BRCA1/2 Mutations, and Elevated Tumor Mutational Burden in Colorectal Cancer".

[This corrects the article DOI: 10.1155/2020/6421205.].

Mucinous Histology, BRCA1/2 Mutations, and Elevated Tumor Mutational Burden in Colorectal Cancer.

Mucinous colorectal carcinomas (MC) constitute 10% of colorectal malignancies. Recently, an increased risk of colorectal cancer has been demonstrated in germline BRCA1/2 mutation carriers. Furthermore, BRCA1/2 germline mutation carriers have exhibited a higher-than-expected frequency of MC tumors. Here, we investigate the relationship between BRCA mutations and mucinous histology in colorectal carcinoma patients, using both an existing cohort of sequenced colorectal tumors and a prospective case-control study comparing MC and conventional adenocarcinoma (AC) patients tested for BRCA mutations. We discovered that MC tumors exhibit a statistically significantly higher incidence of BRCA mutations in addition to a higher average mutation count when compared to AC tumors in the existing cohort. The strongest predictor of the mutation count was mucinous histology, independently of other variables including microsatellite instability. Contrary to our hypothesis, the first association did not recur in the prospective case-control study, likely due to our pathological definition of MC tumors and small sample size. Finally, we observed a higher tumor mutational burden (TMB) in MC tumors compared with AC tumors. We suggest that the association between MC histology, BRCA mutations, and increased TMB may open the door to the utilization of simple tests (such as histopathologic characterization) to detect patients who may benefit from immunotherapy in colorectal cancer.

Sidedness Matters: Surrogate Biomarkers Prognosticate Colorectal Cancer upon Anatomic Location.

BACKGROUND: Anatomic location of primary tumors across the colon correlate with survival in the metastatic setting, whereas left-sided tumors may exhibit superior survival compared with right-sided tumors. The Oncotype Recurrence Score (RS) assay is a clinically validated predictor of recurrence risk in patients with stage II colorectal cancer (CRC). Previous studies had indicated that without adjuvant chemotherapy, CDX2-negative stage II CRC tumors are associated with a lower rate of disease-free survival than CDX2-positive stage II CRC tumors. We aimed to evaluate whether these two validated prognostic biomarkers may correlate with primary tumor location, and whether tumor location may reflect differential prognosis in stage II CRC. MATERIALS AND METHODS: We retrospectively analyzed patients with T3 mismatch repair-proficient (MMR-P) stage II CRC for whom RS assay was performed. Pathological report was reviewed for exact primary tumor location and CDX2 immunostaining. RS and CDX2 expression were correlated with primary tumor location. RESULTS: The analysis included 1,147 patients with MMR-P stage II CRC (median age 69 years [range 29-93]). Tumor distribution across the colon was as follows: 46% (n = 551) were right-sided and 54% (n = 596) were left-sided. RS was higher in right-sided tumors (p = .01). The RS results gradually decreased across the colon (cecum, highest score; sigmoid, lowest score; p = .04). Right-sided tumors exhibited more CDX2-negative tumors (p = .07). CONCLUSION: Our study indicates that right-sided colorectal tumors may display worse prognosis compared with left-sided tumors in MMR-P stage II CRC. Primary tumor location may serve as a prognostic factor that should be taken into account for recurrence risk assessment and consideration of adjuvant treatment. IMPLICATIONS FOR PRACTICE: Sidedness matters, even in stage II colorectal cancer (CRC). Using two previously established prognostic tools, the Oncotype DX assay and CDX2 expression, this study found that right-sided tumors may display worse prognosis compared with left-sided tumors in mismatch repair-proficient stage II CRC. Therefore, primary tumor location should be taken into account for recurrence risk assessment and consideration of adjuvant treatment.

Beyond KRAS: Practical Molecular Targets in Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) has a grim prognosis. Molecular and genomic analyses revealed that the striking majority of these tumors are driven by KRAS mutation, currently not amenable to targeted therapy. However, other driver mutations were found in a small fraction of patients. Herein we report of 3 cases of patients with metastatic PDAC and wildtype KRAS, found to harbor BRAF or RET pathogenic alterations. The patients were treated with targeted therapies with variable success. In our opinion, those proof-of-concept cases argue in favor of additional research and clinical trials' effort in this small but significant PDAC population with uncommon driver mutations.

Topical doxycycline foam 4% for prophylactic management of epidermal growth factor receptor inhibitor skin toxicity: an exploratory phase 2, randomized, double-blind clinical study.

PURPOSE: Acneiform rash, a common toxicity of epidermal growth factor receptor inhibitors (EGFRIs), can cause patient discomfort, warranting changes in treatment. This study investigated the safety, tolerability, and efficacy of a novel doxycycline foam, FDX104 4%, for managing EGFRI-related skin toxicity. METHODS: This was an exploratory phase 2, randomized, double-blind, placebo-controlled study. Subjects had metastatic colorectal cancer and were being treated with either cetuximab or panitumumab plus chemotherapy. Treatment (twice-daily topical FDX104 4% on one side of the face and vehicle foam on the other for 5 weeks) was initiated 7 ± 3 days prior to EGFRI therapy. Rash severity, safety, and tolerability were evaluated at 2 and 4 weeks after EGFRI start. RESULTS: The mean maximal rash grade was lower with FDX104 4% vs vehicle, and fewer subjects developed moderate-to-severe (grades 2-3) rash. On the Global Severity Score scale, a statistically significant difference favored FDX104 4% over vehicle (P = .047). Adverse events (AEs) (n = 68) occurred in 20 subjects; most were mild or moderate. The most common AEs were oral mucositis, nausea, and vomiting, common to chemotherapy and EGFRI treatment. Study-drug-related AEs were experienced by five subjects and consisted of mild, local skin reactions. No study-drug-related systemic side effects were reported. CONCLUSION: Twice-daily, topical administration of FDX104 4% as an adjunct to either cetuximab or panitumumab was safe and well tolerated, and appeared to prevent the onset of rash, especially severe rash. CLINICALTRIALS. GOV IDENTIFIER: Trial Registration NCT02239731.

Defects in homologous recombination repair genes are associated with good prognosis and clinical sensitivity to DNA-damaging agents in pancreatic cancer: A case report.

Tumor genome sequencing is important for increasing our understanding of the development of cancer, which may be affected by different therapies. In the present study, genomic evolution was investigated in a patient with stage IV pancreatic cancer bearing a germline breast cancer 2 (BRCA2) mutation. The patient received cisplatin, a DNA cross-linking agent, which led to a long-lasting complete response. Eventually the patient developed brain metastasis, suggesting the acquisition of resistance to cisplatin. He subsequently underwent brain lesion resection, radiofrequency ablation and chemotherapy, again resulting in long-lasting response. Samples of blood, pancreatic tumor tissue and brain metastases were collected and the extracted DNA was sequenced. The pancreatic and brain lesions, when compared with the blood samples, exhibited mutations in the BRCA1 and checkpoint kinase 2 genes, in addition to the germline BRCA2 mutation. The brain lesion, when compared with the primary tumor, harbored no additional mutations or copy-number variations. These findings suggest that the isolated relapse in the brain was due to pharmacological sanctuary rather than genomic alterations. It may be suggested that the presence of defects in the homologous recombination repair pathways are associated with a good prognosis and clinical sensitivity to agents that damage the DNA in pancreatic cancer.

Phenotypic characteristics of colorectal cancer in BRCA1/2 mutation carriers.

Mutations in the BRCA1/2 genes were recently shown to be associated with an increased risk for colorectal cancer. We characterized the largest cohort available of BRCA1/2 mutation carriers with colorectal cancer. We analyzed 32 patients with lower gastrointestinal cancers and germline BRCA1/2 mutations from two large academic hospital registries; 91% of patients were of Ashkenazi ancestry, 78% were women, and 62.5% were carriers of BRCA1 gene mutations. A high percentage of colorectal tumors (34.5%) had a mucinous histology and were located atypically in the left colon. Two patients had anal cancer with unusual histology and an additional patient had mucinous small bowel carcinoma. Gene expression analysis showed significant correlation between the gene signatures of left mucinous colorectal cancer and basal-like breast cancer. Our results imply that Ashkenazi BRCA1/2 mutation carriers with colorectal cancer might have unique characteristics with a high rate of left-sided, mucinous histology colorectal cancer, and possibly anal carcinoma. This report suggests a phenotypic influence of defects in DNA repair genes on colorectal tumors.