Progressive multifocal leukoencephalopathy associated with chemotherapy induced lymphocytopenia in solid tumors – case report of an underestimated complication

Background JC virus reactivation causing progressive multifocal leukoencephalopathy (PML) occurs preferentially in human immunodeficiency virus (HIV) positive individuals or patients suffering from hematologic neoplasms due to impaired viral control. Reactivation in patients suffering from solid malignancies is rarely described in published literature. Case Presentation Here we describe a case of PML in a male patient suffering from esophageal cancer who underwent neoadjuvant radiochemotherapy and surgical resection in curative intent resulting in complete tumor remission. The radiochemotherapy regimen contained carboplatin and paclitaxel (CROSS protocol). Since therapy onset, the patient presented with persistent and progredient leukopenia and lymphopenia in absence of otherwise known risk factors for PML. Symptom onset, which comprised aphasia, word finding disorder, and paresis, was apparent 7 months after therapy initiation. There was no relief in symptoms despite standard of care PML directed supportive therapy. The patient died two months after therapy onset. Conclusion PML is a very rare event in solid tumors without obvious states of immununosuppression and thus harbors the risk of unawareness. The reported patient suffered from lymphopenia, associated with systemic therapy, but was an otherwise immunocompetent individual. In case of neurologic impairment in patients suffering from leukopenia, PML must be considered – even in the absence of hematologic neoplasia or HIV infection

Changes of microrna levels in plasma of patients with rectal cancer during chemoradiotherapy

Since the response to chemoradiotherapy in patients with locally advanced rectal cancer is heterogeneous, valid biomarkers are needed to monitor tumor response. Circulating microRNAs are promising candidates, however analyses of circulating microRNAs in rectal cancer are still rare. 111 patients with rectal cancer and 46 age-matched normal controls were enrolled. The expression levels of 30 microRNAs were analyzed in 17 pre-treatment patients’ plasma samples. Differentially regulated microRNAs were validated in 94 independent patients. For 52 of the 94 patients a paired comparison between pre-treatment and post-treatment samples was performed. miR-17, miR-18b, miR-20a, miR-31, and miR-193a_3p, were significantly downregulated in pre-treatment plasma samples of patients with rectal cancer (p < 0.05). miR-29c, miR-30c, and miR-195 showed a trend of differential regulation. After validation, miR-31 and miR-30c were significantly deregulated by a decrease of expression. In 52 patients expression analyses of the 8 microRNAs in matched pre-treatment and post-treatment samples showed a significant decrease for all microRNAs (p < 0.05) after treatment. Expression levels of miR-31 and miR-30c could serve as valid biomarkers if validated in a prospective study. Plasma microRNA expression levels do not necessarily represent miRNA expression levels in tumor tissue. Also, expression levels of microRNAs change during multimodal therapy

Aberrant DNA methylation patterns in microsatellite stable human colorectal cancers define a new marker panel for the CpG island methylator phenotype

A distinct group of colorectal carcinomas (CRCs) referred to as the “CpG island methylator phenotype” (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status

Cytosolic 5'-nucleotidase 1A is overexpressed in pancreatic cancer and mediates gemcitabine resistance by reducing intracellular gemcitabine metabolites.

BACKGROUND: Cytosolic 5'-nucleotidase 1A (NT5C1A) dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. Here, we investigate NT5C1A expression in pancreatic ductal adenocarcinoma (PDAC) and its impact on gemcitabine metabolism and therapeutic efficacy. METHODS: NT5C1A expression was determined by semiquantitative immunohistochemistry using tissue microarrays. Gemcitabine metabolites and response were assessed in several human and murine PDAC cell lines using crystal violet assays, Western blot, viability assays, and liquid chromatography tandem mass-spectrometry (LC-MS/MS). FINDINGS: NT5C1A was strongly expressed in tumor cells of a large subgroup of resected PDAC patients in two independent patient cohorts (44-56% score 2 and 8-26% score 3, n = 414). In contrast, NT5C1A was expressed at very low levels in the tumor stroma, and neither stromal nor tumoral expression was a prognostic marker for postoperative survival. In vitro, NT5C1A overexpression increased gemcitabine resistance by reducing apoptosis levels and significantly decreased intracellular amounts of cytotoxic dFdCTP in +NT5C1A tumor cells. Co-culture experiments with conditioned media from +NT5C1A PSCs improved gemcitabine efficacy in tumor cells. In vivo, therapeutic efficacy of gemcitabine was significantly decreased and serum levels of the inactive gemcitabine metabolite dFdU significantly increased in mice bearing NT5C1A overexpressing tumors. INTERPRETATION: NT5C1A is robustly expressed in tumor cells of resected PDAC patients. Moreover, NT5C1A mediates gemcitabine resistance by decreasing the amount of intracellular dFdCTP, leading to reduced tumor cell apoptosis and larger pancreatic tumors in mice. Further studies should clarify the role of NT5C1A as novel predictor for gemcitabine treatment response in patients with PDAC

Impact of Endoscopic Vacuum Therapy with Low Negative Pressure for Esophageal Perforations and Postoperative Anastomotic Esophageal Leaks

Introduction: Management of esophageal anastomotic leaks (AL) and esophageal perforations (EP) remains difficult and often requires an interdisciplinary treatment modality. For primary endoscopic management, self-expanding metallic stent (SEMS) placement is often considered first-line therapy. Recently, endoscopic vacuum therapy (EVT) has emerged as an alternative or adjunct for management of these conditions. So far, data for EVT in the upper gastrointestinal-tract is restricted to single centre, non-randomized trials. No studies on optimal negative pressure application during EVT exist. The aim of our study is to describe our centre’s experience with low negative pressure (LNP) EVT for these indications over the past 5-years. Patients and Methods: Between January 2014 and December 2018, 30 patients were endoscopically treated for AL (n = 23) or EP (n = 7). All patients were primarily treated with EVT and LNP between –20 and –50 mm Hg. Additional endoscopic treatment was added when EVT failed. Procedural and peri-procedural data, as well as clinical outcomes including morbidity and mortality, were analysed. Results: Clinical successful endoscopic treatment of EP and AL was achieved in 83.3% (n = 25/30), with 73.3% success using EVT alone (n = 22/30). Mean treatment duration until leak closure was 16.1 days (range 2–58 days). Additional treatment modalities for complete leak resolution was necessary in 10% (n = 3/30), including SEMS placement and fibrin glue injection. Mean hospital stay for patients with EP was shorter with 33.7 days compared to AL with 54.4 days (p = 0.08). Estimated preoperative 10-year overall survival (Charlson comorbidity score) was 39.4% in patients with AL and 59.9% in patients with EP (p = 0.26). A mean of 5.1 EVT changes (range 1–12) was needed in EP and 3.6 changes (range 1–13) in AL to achieve complete closure, switch to other treatment modality, or reach endoscopic failure (p = 0.38). Conclusion: LNP EVT enables effective minimally – invasive endoluminal leak closure from anastomotic esophageal leaks and EP in high-morbid patients. In this study, EVT was combined with other endoscopic treatment options such as SEMS placement or fibrin glue injection in order to achieve leak or perforation closure in the vast majority of patients (83.3%). Low aspiration pressures led to slower but still sufficient clinical results

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5' end resection near the fork junction, which permits 3' single strand invasion of a homologous template for fork restart. This 5' end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5' DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5' overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ

Impact of RNA degradation on gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.</p> <p>Methods</p> <p>To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.</p> <p>Results</p> <p>The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.</p> <p>Conclusions</p> <p>Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.</p

DPEP1 Inhibits Tumor Cell Invasiveness, Enhances Chemosensitivity and Predicts Clinical Outcome in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. To identify biologically relevant genes with prognostic and therapeutic significance in PDAC, we first performed the microarray gene-expression profiling in 45 matching pairs of tumor and adjacent non-tumor tissues from resected PDAC cases. We identified 36 genes that were associated with patient outcome and also differentially expressed in tumors as compared with adjacent non-tumor tissues in microarray analysis. Further evaluation in an independent validation cohort (N = 27) confirmed that DPEP1 (dipeptidase 1) expression was decreased (T: N ratio ∼0.1, P<0.01) in tumors as compared with non-tumor tissues. DPEP1 gene expression was negatively correlated with histological grade (Spearman correlation coefficient = −0.35, P = 0.004). Lower expression of DPEP1 in tumors was associated with poor survival (Kaplan Meier log rank) in both test cohort (P = 0.035) and validation cohort (P = 0.016). DPEP1 expression was independently associated with cancer-specific mortality when adjusted for tumor stage and resection margin status in both univariate (hazard ratio = 0.43, 95%CI = 0.24–0.76, P = 0.004) and multivariate analyses (hazard ratio = 0.51, 95%CI = 0.27–0.94, P = 0.032). We further demonstrated that overexpression of DPEP1 suppressed tumor cells invasiveness and increased sensitivity to chemotherapeutic agent Gemcitabine. Our data also showed that growth factor EGF treatment decreased DPEP1 expression and MEK1/2 inhibitor AZD6244 increased DPEP1 expression in vitro, indicating a potential mechanism for DPEP1 gene regulation. Therefore, we provide evidence that DPEP1 plays a role in pancreatic cancer aggressiveness and predicts outcome in patients with resected PDAC. In view of these findings, we propose that DPEP1 may be a candidate target in PDAC for designing improved treatments