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 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.

[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.

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.

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.

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.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

A phase 1/2a, dose-escalation, safety, pharmacokinetic, and preliminary efficacy study of intraperitoneal administration of BC-819 (H19-DTA) in subjects with recurrent ovarian/peritoneal cancer.

BACKGROUND: H19 is a paternally imprinted, oncofetal gene expressed in various embryonic tissues and in 85% of the ovarian tumors. H19-DTA (BC-819) is a DNA plasmid that drives the expression of the diphtheria toxin gene under the regulation of the H19 promoter sequence and therefore is a potential treatment for various tumors that overexpress the H19 gene, among them-ovarian cancer. OBJECTIVE: To assess the safety and efficacy of intra-peritoneal (IP) instillations of H19-DTA (BC-819) plasmid in treating ovarian/peritoneal cancer patients with advanced recurrent disease. METHODS: A phase 1-2A multi-centric trial included 14 eligible patients who were either platinum-refractory or platinum-resistant with positive H19 expression. Patients were treated IP with escalating weekly doses of BC-819 for a maximum of 6-9 weeks. Dose-limiting toxicities (DLT) were assessed after the first course of treatment for each patient and each subsequent cohort was enrolled once each subject had completed the first course of treatment and its 4-week follow-up period. The occurrence of adverse events (AEs) and response to treatment were assessed after the induction course and then periodically. RESULTS: During the study, no DLTs were observed. Only 5 grade 1 and 2 AEs, which occurred in 4 patients were considered as possibly related to BC-819. The best tumor response seen was stable disease. Median survivals of 3.2, 5.3 and 6.5 months were observed for the 60, 120 and 240 mg cohorts, respectively. CONCLUSIONS: BC-819 can be considered safe and well tolerated in intraperitoneal doses up to 240 mg. Hybridization of intraperitoneal chemotherapy with the biological treatment of BC-819 should be further evaluated in phase 2 and 3 studies.

Screening for germline mutations in breast/ovarian cancer susceptibility genes in high-risk families in Israel.

We evaluated the clinical utility of screening for mutations in 34 breast/ovarian cancer susceptibility genes in high-risk families in Israel. Participants were recruited from 12, 2012 to 6, 2015 from 8 medical centers. All participants had high breast/ovarian cancer risk based on personal and family history. Genotyping was performed with the InVitae™ platform. The study was approved by the ethics committees of the participating centers; all participants gave a written informed consent before entering the study. Overall, 282 individuals participated in the study: 149 (53 %) of Ashkenazi descent, 80 (28 %) Jewish non-Ashkenazi descent, 22 (8 %) of mixed Ashkenazi/non-Ashkenazi origin, 21 (7 %) were non-Jewish Caucasians, and the remaining patients (n = 10-3.5 %) were of Christian Arabs/Druze/unknown ethnicity. For breast cancer patients (n = 165), the median (range) age at diagnosis was 46 (22-90) years and for ovarian cancer (n = 15) 54 (38-69) years. Overall, 30 cases (10.6 %) were found to carry a pathogenic actionable mutation in the tested genes: 10 BRCA1 (3 non-founder mutations), 9 BRCA2 (8 non-founder mutations), and one each in the RAD51C and CHEK2 genes. Furthermore, actionable mutations were detected in 9 more cases in 4 additional genes (MSH2, RET, MSH6, and APC). No pathogenic mutations were detected in the other genotyped genes. In this high-risk population, 10.6 % harbored an actionable pathogenic mutation, including non-founder mutations in BRCA1/2 and in additional cancer susceptibility genes, suggesting that high-risk families should be genotyped and be assigned a genotype-based cancer risk.

Efficacy and safety of olaparib monotherapy in germline BRCA1/2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy.

OBJECTIVE: The efficacy and safety of olaparib, an oral poly(ADP-ribose) polymerase (PARP) inhibitor, was investigated in a subgroup of patients with germline BRCA1/2 mutated (gBRCA1/2m) advanced ovarian cancer who had received ≥3 prior lines of chemotherapy. Primary data from this Phase II study (Study 42, ClinicalTrials.govNCT01078662) have been reported previously. METHODS: Eligible patients were treated with oral olaparib 400mg bid capsule monotherapy until disease progression according to RECIST v1.1. Objective response rate (ORR) and duration of response (DoR) were assessed for patients with measurable disease at baseline. Safety and tolerability were assessed for all patients by adverse event (AE) incidence and changes in laboratory parameters. Platinum resistance status was obtained retrospectively, and responses to olaparib evaluated. RESULTS: In patients with gBRCA1/2m ovarian cancer, 154/193 (80%) had received ≥3 prior lines of chemotherapy, of whom 137/154 (89%) had measurable disease at baseline. ORR was 34% (46/137; 95% confidence interval [CI] 26-42) and median DoR was 7.9 (95% CI 5.6-9.6) months. ORR in platinum-resistant tumors was 30%. Median DoR for platinum-sensitive and platinum-resistant disease was similar: 8.2months (95% CI 5.6-13.5) compared with 8.0months (4.8-14.8), respectively. Six of the 193 (3%) patients had an AE with an outcome of death. None of these AEs at time of occurrence was considered causally related to olaparib. CONCLUSION: Following ≥3 prior lines of chemotherapy, olaparib 400mg bid (capsule form) monotherapy demonstrated notable antitumor activity in patients with gBRCA1/2m advanced ovarian cancer. No new safety signals were identified.

Impact of the 12-Gene Colon Cancer Assay on Clinical Decision Making for Adjuvant Therapy in Stage II Colon Cancer Patients.

OBJECTIVES: To evaluate the impact of the 12-gene Colon Cancer Recurrence Score Assay-a clinically validated prognosticator in stage II colon cancer after surgical resection-on adjuvant treatment decisions in T3 mismatch repair proficient (MMR-P) stage II colon cancer in clinical practice. METHODS: This retrospective analysis included all patients with T3 MMR-P stage II colon cancer (Clalit Health Services members) with Recurrence Score results (time frame January 2011 to May 2012). Treatment recommendations pretesting were compared with the treatments received. Changes were categorized as decreased (to observation alone/removing oxaliplatin from the therapy) or increased (from observation alone/adding oxaliplatin to the therapy) intensity. RESULTS: The analysis included 269 patients; 58%, 32%, and 10% of the values were in the low (<30), intermediate (30-40), and high (≥41) score groups, respectively. In 102 patients (38%), treatment changed post-testing (decreased/increased intensity 76/26 patients). The overall impact was decreased chemotherapy use (45.0% to 27.9%; P < 0.001). Treatment changes occurred in all score groups, but more frequently in the high (change rate 63.0%; 95% confidence interval [CI] 42.3%-80.6%) than in the intermediate (30.6%; 95% CI 21.0%-41.5%) and low (37.6%; 95% CI 30.0%-45.7%) score groups. The direction of the change was consistent with the assay result, with increased intensity more common in higher score values and decreased intensity more common in lower score values. CONCLUSIONS: Testing significantly affected adjuvant treatment in T3 MMR-P stage II colon cancer in clinical practice. The study is limited by its design, which compared treatment recommendations pretesting to actual treatments received post-testing, lack of a control group, and nonassessment of confounding factors that may have affected treatment decisions.

Mutant KRAS is a druggable target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial-mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.

Oncofetal H19 RNA promotes tumor metastasis.

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-ß and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-ß treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

Retrospective Study of the Predictive Value of Target Now in Systemic Therapy for Metastatic Colorectal and Gastric Carcinomas.

BACKGROUND: Predictive biomarkers for personalized treatment of neoplasms are suggested to be a major advancement in oncology and are increasingly used in clinical practice, albeit based on level II evidence. Target Now (TN) employs immunostaining and RNA expression on tumor samples to identify potentially beneficial or ineffective drugs. OBJECTIVES: To explore retrospectively the predictive value of TN for patients with colorectal and gastric carcinomas. METHODS: The study group comprised colorectal and gastric carcinoma patients with TN test reports. We identified chemotherapy regimens given for stage IV disease for which TN reports indicated prediction. Protocols were classified as having clinical benefit (CB; i.e., stable disease or any objective response) or progressive disease, and this was compared with the TN prediction. RESULTS: Nineteen patients--12 colorectal and 7 gastric carcinomas--met the inclusion criteria. There were 26 evaluable treatment protocols; of 18 with a CB15 were predicted to have a CB while 3 were predicted to have a lack of CB. Of eight protocols that had no CB, seven were predicted to have a CB and one a lack of CB. A chi-square test was non-significant (P = 0.78). An exploratory analysis yielded a positive predictive value of 68% and a sensitivity of 83% for the TN test. CONCLUSIONS: This study emphasizes the need for larger multi-center studies to validate the TN test before it is adopted into clinical practice.

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.

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.

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.

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.

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.