Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients’ informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort

Non-professional phagocytosis: a general feature of normal tissue cells

Non-professional phagocytosis by cancer cells has been described for decades. Recently, non-professional phagocytosis by normal tissue cells has been reported, which prompted us to take a closer look at this phenomenon. Non-professional phagocytosis was studied by staining cultured cells with live-cell staining dyes or by staining paraffin-embedded tissues by immunohistochemistry. Here, we report that each of 21 normal tissue cell lines from seven different organs was capable of phagocytosis, including ex vivo cell cultures examined before the 3rd passage as well as the primary and virus-transformed cell lines. We extended our analysis to an in vivo setting, and we found the occurrence of non-professional phagocytosis in healthy skin biopsies immediately after resection. Using dystrophin immunohistochemistry for membrane staining, human post-infarction myocardial tissue was assessed. We found prominent signs of non-professional phagocytosis at the transition zone of healthy and infarcted myocardia. Taken together, our findings suggest that non-professional phagocytosis is a general feature of normal tissue cells

One Step Nucleic Acid Amplification (OSNA) - a new method for lymph node staging in colorectal carcinomas

<p>Abstract</p> <p>Background</p> <p>Accurate histopathological evaluation of resected lymph nodes (LN) is essential for the reliable staging of colorectal carcinomas (CRC). With conventional sectioning and staining techniques usually only parts of the LN are examined which might lead to incorrect tumor staging. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and therefore improve staging.</p> <p>Methods</p> <p>OSNA is based on a short homogenisation step and subsequent automated amplification of cytokeratin 19 (CK19) mRNA directly from the sample lysate, with result available in 30-40 minutes. In this study 184 frozen LN from 184 patients with CRC were investigated by both OSNA and histology (Haematoxylin & Eosin staining and CK19 immunohistochemistry), with half of the LN used for each method. Samples with discordant results were further analysed by RT-PCR for CK19 and carcinoembryonic antigen (CEA).</p> <p>Results</p> <p>The concordance rate between histology and OSNA was 95.7%. Three LN were histology+/OSNA- and 5 LN histology-/OSNA+. RT-PCR supported the OSNA result in 3 discordant cases, suggesting that metastases were exclusively located in either the tissue analysed by OSNA or the tissue used for histology. If these samples were excluded the concordance was 97.2%, the sensitivity 94.9%, and the specificity 97.9%. Three patients (3%) staged as UICC I or II by routine histopathology were upstaged as LN positive by OSNA. One of these patients developed distant metastases (DMS) during follow up.</p> <p>Conclusion</p> <p>OSNA is a new and reliable method for molecular staging of lymphatic metastases in CRC and enables the examination of whole LN. It can be applied as a rapid diagnostic tool to estimate tumour involvement in LN during the staging of CRC.</p

Protein tyrosine phosphatase non-receptor type 2 controls colorectal cancer development

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC) as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. Particularly, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T-cells and increased PTPN2 levels negatively correlated with PD1, CTLA4, STAT1 and granzyme A. In vivo, T-cell and dendritic cell-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44+ effector/memory T-cells, as well as CD8+ T-cell infiltration and cytotoxicity into the tumor. In direct relevance to CRC treatment, T-cell-specific PTPN2 deletion potentiated anti-PD-1 efficacy and induced anti-tumor memory formation upon tumor re-challenge in vivo. Our data suggest a role for PTPN2 in suppressing anti-tumor immunity and promoting tumor development in CRC patients. Our in vivo results uncover PTPN2 as a key player in controlling immunogenicity of CRC, with the strong potential to be exploited to promote cancer immunotherapy

N-glycosylation regulates intrinsic IFN-γ resistance in colorectal cancer: implications for immunotherapy

Background &amp; Aims: Advanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. Methods: Spontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. Results: The conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ–resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling. Conclusions: Together, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC

Distal colonic atresia: a case report

Colonic atresia (CA) is a rare disease with an incidence range between one of 20 000 and one of 66 000 live births. Most CA are located within the proximal colon; distal CA are even rarer. Because of its rarity, another case shall be described herewith. A 37th week of pregnancy born child was noticed occurring multiple vomiting, a distended abdomen and additional whitish-bloody stool shortly thereafter. In the first operation, a double-barrel stoma was created. After sufficient weight gain and alignment of the stoma ends, a secondary anastomosis was created in the child after 2 months. The diagnosis can be made reliably on the basis of an X-ray and leads to a good outcome with prompt surgical intervention. However, accompanying malformations should always be considered

Cholelithiasis and cholecystitis in children and adolescents: Does this increasing diagnosis require a common guideline for pediatricians and pediatric surgeons?

Background!#!In contrast to adults, for whom guidelines on the cholelithiasis treatment exist, there is no consistent treatment of pediatric patients with cholelithiasis throughout national and international departments, most probably due to the lack of evidence-based studies.!##!Methods!#!We evaluated the German management of pediatric cholelithiasis in a dual approach. Firstly, a retrospective, inter-divisional study was established, comparing diagnostics and therapy of patients of the pediatric surgery department with the management of patients aged &amp;lt; 25 years of the visceral surgery department in our institution over the past ten years. Secondarily, a nation-wide online survey was implemented through the German Society of Pediatric Surgery.!##!Results!#!Management of pediatric patients with cholelithiasis was primarily performed by pediatricians in the retrospective analysis (p &amp;lt; 0.001). Pediatric complicated cholelithiasis was not managed acutely in the majority of cases with a median time between diagnosis and surgery of 22 days (range 4 days-8 months vs. 3 days in visceral surgery subgroup (range 0 days-10 months), p = 0.003). However, the outcome remained comparable. The hospital's own results triggered a nation-wide survey with a response rate of 38%. Primary pediatric medical management of patients was confirmed by 36 respondents (71%). In case of acute cholecystitis, 22% of participants perform a cholecystectomy within 24 h after diagnosis. Open questions revealed that complicated cholelithiasis is managed individually.!##!Conclusions!#!The management of pediatric cholelithiasis differs between various hospitals and between pediatricians and pediatric surgeons. Evidence-based large-scale population studies as well as a common guideline may represent very important tools for treating this increasing diagnosis

A Pilot Study of Non-invasive Sacral Nerve Stimulation in Treatment of Constipation in Childhood and Adolescence

Background/Aims: Constipation shows both, a high prevalence and a significant impact. However, it is often perceived as minor and treatment choices are limited. The neuromodulation approach is a valuable option to be considered. This study assesses the use of non-invasive sacral nerve stimulation to reduce constipation in children. Methods: Between February 2013 and May 2015, pediatric patients with chronic constipation were treated with this non-invasive neuromodulation procedure, adapted from classical sacral nerve stimulation. A stimulation device attached to adhesive electrodes on the lower abdomen and back generated an electrical field with a stable frequency of 15 Hz via variable stimulation intensity (1–10 V). The effect of therapy was evaluated in routine check-ups and by specialized questionnaires. Results: The study assessed non-invasive sacral nerve stimulation in 17 patients (9 boys, 8 girls, mean age 6.5 years). They underwent stimulation with 6–9 V for a mean of 11 h per day (range 0.5–24 h) over a mean of 12.7 weeks. Improvement of constipation was achieved in more than half of the patients (12/17) and sustained in almost half of these patients (5/12). Complications were minor (skin irritation, electrode dislocation). Conclusions: Non-invasive sacral nerve stimulation appears to be effective in achieving improvement in pediatric patients with chronic constipation. As an additional external neuromodulation concept, this stimulation may represent a relevant addition to currently available therapeutic options. Further studies are needed to confirm these results