ZEB1-mediated fibroblast polarization controls inflammation and sensitivity to immunotherapy in colorectal cancer.

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

Intestinal Barrier Healing Is Superior to Endoscopic and Histologic Remission for Predicting Major Adverse Outcomes in Inflammatory Bowel Disease: The Prospective ERIca Trial.

BACKGROUND & AIMS: Endoscopic and histologic remission have emerged as key therapeutic goals in the management of inflammatory bowel diseases (IBD) that are associated with favorable long-term disease outcomes. Here, we prospectively compared the predictive value of barrier healing with endoscopic and histologic remission for predicting long-term disease behavior in a large cohort of patients with IBD in clinical remission. METHODS: At baseline, patients with IBD in clinical remission underwent ileocolonoscopy with assessment of intestinal barrier function by confocal endomicroscopy. Endoscopic and histologic disease activity, as well as barrier healing, was prospectively assessed along established scores. During subsequent follow-up, patients were closely monitored for clinical disease activity and the occurrence of major adverse outcomes (MAOs): disease flares, IBD-related hospitalization or surgery, and initiation or dose escalation of systemic steroids, immunosuppressants, small molecules, or biological therapy. RESULTS: The final analysis included 181 patients, 100 with Crohn's disease [CD] and 81 with ulcerative colitis (UC). During a mean follow-up of 35 (CD) and 25 (UC) months, 73% of patients with CD and 69% of patients with UC experienced at least 1 MAO. The probability of MAO-free survival was significantly higher in patients with IBD with endoscopic remission compared with endoscopically active disease. In addition, histologic remission predicted MAO-free survival in patients with UC but not CD. Barrier healing on endomicroscopy was superior to endoscopic and histologic remission for predicting MAO-free survival in both UC and CD. CONCLUSIONS: Barrier healing is associated with decreased risk of disease progression in patients with clinically remittent IBD, with superior predictive performance compared with endoscopic and histologic remission. Analysis of barrier function might be considered as a future treatment target in clinical trials. CLINICALTRIALS: gov number, NCT05157750.

Associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 with molecular subtypes, PD-L1 expression, and FGFR3 mutational status in two advanced urothelial bladder cancer cohorts.

AIMS: Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody-drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN-4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. METHODS AND RESULTS: The TCGA BLCA (n = 405) and the CCC-EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN-4/NECTIN-4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN-4/NECTIN-4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN-4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H-score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN-4 expression was reduced in neuroendocrine-like and/or protein-based double-negative tumours. TROP2- and NECTIN-4-negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD-L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN-4 expression. CONCLUSIONS: TACSTD2/TROP2 and NECTIN-4/NECTIN-4 are widely expressed in aUC, independent of FGFR3 alterations or PD-L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(-like) and sarcomatoid aUC.

The Immune Checkpoint BTLA in Oral Cancer: Expression Analysis and Its Correlation to Other Immune Modulators.

In oral squamous cell carcinoma (OSCC) tissues, an immunotolerant situation triggered by immune checkpoints (ICPs) can be observed. Immune checkpoint inhibitors (ICIs) against the PD1/PD-L axis are used with impressive success. However, the response rate is low and the development of acquired resistance to ICI treatment can be observed. Therefore, new treatment strategies especially involving immunological combination therapies need to be developed. The novel negative immune checkpoint BTLA has been suggested as a potential biomarker and target for antibody-based immunotherapy. Moreover, improved response rates could be displayed for tumor patients when antibodies directed against BTLA were used in combination with anti-PD1/PD-L1 therapies. The aim of the study was to check whether the immune checkpoint BTLA is overexpressed in OSCC tissues compared to healthy oral mucosa (NOM) and could be a potential diagnostic biomarker and immunological target in OSCC. In addition, correlation analyses with the expression of other checkpoints should clarify more precisely whether combination therapies are potentially useful for the treatment of OSCC. A total of 207 tissue samples divided into 2 groups were included in the study. The test group comprised 102 tissue samples of OSCC. Oral mucosal tissue from 105 healthy volunteers (NOM) served as the control group. The expression of two isoforms of BTLA (BTLA-1/2), as well as PD1, PD-L1/2 and CD96 was analyzed by RT-qPCR. Additionally, BTLA and CD96 proteins were detected by IHC. Expression levels were compared between the two groups, the relative differences were calculated, and statistical relevance was determined. Furthermore, the expression rates of the immune checkpoints were correlated to each other. BTLA expression was significantly increased in OSCC compared to NOM (pBTLA_1 = 0.003; pBTLA_2 = 0.0001, pIHC = 0.003). The expression of PD1, its ligands PD-L1 and PD-L2, as well as CD96, were also significantly increased in OSCC (p ≤ 0.001). There was a strong positive correlation between BTLA expression and that of the other checkpoints (p < 0.001; ρ ≥ 0.5). BTLA is overexpressed in OSCC and appears to be a relevant local immune checkpoint in OSCC. Thus, antibodies directed against BTLA could be potential candidates for immunotherapies, especially in combination with ICI against the PD1/PD-L axis and CD96.

ATF2 loss promotes 5-FU resistance in colon cancer cells via activation of the ATR-Chk1 damage response pathway.

BACKGROUND: The role of ATF2 in colon cancer (CC) is controversial. Recently, we reported that low ATF2 expression is characteristic of highly invasive tumors, suggesting that ATF2 might also be involved in therapy resistance. 5-Fluorouracil (5-FU) is the best-known chemotherapeutic drug for CC, but drug resistance affects its curative effect. To date, the role of ATF2 in the 5-FU response remains elusive. METHODS/RESULTS: For our study, we had available HCT116 cells (wild-type p53) and HT29 colon tumor cells (mutant p53) and their corresponding CRISPR‒Cas9-generated ATF2-KO clones. We observed that loss of ATF2 triggered dose- and time-dependent 5-FU resistance in HCT116 cells by activating the DNA damage response (DDR) pathway with high p-ATRThr1989 and p-Chk1Ser317 levels accompanied by an increase in the DNA damage marker γ-H2AX in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. Chk1 inhibitor studies causally displayed the link between DDR and drug resistance. There were contradictory findings in HT29 ATF2-KO cells upon 5-FU exposure with low p-Chk1Ser317 levels, strong apoptosis induction, but no effects on DNA damage. In ATF2-silenced HCT116 p53-/- cells, 5-FU did not activate the DDR pathway. Co-immunoprecipitation and proximity ligation assays revealed that upon 5-FU treatment, ATF2 binds to ATR to prevent Chk1 phosphorylation. Indeed, in silico modelling showed reduced ATR-Chk1 binding when ATF2 was docked into the complex. CONCLUSIONS: We demonstrated a novel ATF2 scaffold function involved in the DDR pathway. ATF2-negative cells are highly resistant due to effective ATR/Chk1 DNA damage repair. Mutant p53 seems to overwrite the tumor suppressor function of ATF2.

Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer.

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelial-mesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ATF2 loss promotes tumor invasion in colorectal cancer cells via upregulation of cancer driver TROP2.

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

Dynamic study of the finger interphalangeal joint volar plate-motion analysis with magnetic resonance cinematography and histologic comparison.

OBJECTIVE: We aimed to further improve knowledge about volar plate (VP) motion of the finger proximal interphalangeal joint (PIP), by analyzing the dynamic VP shape during a full range of finger flexion using magnetic resonance cinematography of the fingers (MRCF), and to compare the results with anatomical cross sections from cadaver specimens. MATERIALS AND METHODS: The dynamic sagittal VP shape was visualized with MRCF in a total number of 23 healthy volunteers. The length, angle, and thickness as well as the contact length of the VP to the PIP joint base were measured. Statistical analysis included t-test or rank-sum testing. Anatomical cross sections with differing degrees of PIP joint flexion were obtained from 12 cadaver specimens (fingers) for comparison. RESULTS: Significant positive correlations between PIP joint flexion angle and VP area, length, depth and the VP contact length were found. This matched histologically to fiber rearrangements especially within the loose third VP layer. CONCLUSION: Our study analyzed the full range of motion dynamic VP shape of the PIP joint using MRCF. This contributes to a more precise understanding of the complex interaction of the VP with the PIP joint and may facilitate evaluation of clinical cases such as VP avulsion or pulley rupture.

Cervical intraepithelial neoplasia grade 3: development during pregnancy and postpartum.

PURPOSE: The aims of the present study were to evaluate the development of untreated cervical intraepithelial neoplasia (CIN) 3 during pregnancy and to assess persistence, progression, and regression rates postpartum to identify factors associated with regression. METHODS: In a tertiary gynecology and obstetrics department, a total of 154 pregnant women with CIN 3 were treated in the dysplasia unit. The follow-up findings were analyzed retrospectively on the basis of histological, cytological, and human papillomavirus (HPV) testing of 154 pregnant women confirmed as having CIN 3 in colposcopically guided biopsies. RESULTS: The rates of persistence, regression, and progression of CIN 3 in these women were 76.1%, 20% and 3.2%, respectively. Data for the delivery mode was available for 126 women. The rate of regression was almost twice as high with vaginal delivery as with cesarean section, at 27.4 vs. 15.2%, whereas the rate of progression was lower with vaginal delivery, at 2.7 vs. 6.5%. CONCLUSION: The rate of persistence of CIN observed in this study is comparable to that reported in other studies. The study provides strong evidence for greater regression among women who have vaginal deliveries. Careful work-up is recommended postpartum for this group of women in order to rule out persistent CIN 3 or invasive disease.

Colon Cancer Microbiome Landscaping: Differences in Right- and Left-Sided Colon Cancer and a Tumor Microbiome-Ileal Microbiome Association.

In the current era of precision oncology, it is widely acknowledged that CRC is a heterogeneous disease entity. Tumor location (right- or left-sided colon cancer or rectal cancer) is a crucial factor in determining disease progression as well as prognosis and influences disease management. In the last decade, numerous works have reported that the microbiome is an important element of CRC carcinogenesis, progression and therapy response. Owing to the heterogeneous nature of microbiomes, the findings of these studies were inconsistent. The majority of the studies combined colon cancer (CC) and rectal cancer (RC) samples as CRC for analysis. Furthermore, the small intestine, as the major site for immune surveillance in the gut, is understudied compared to the colon. Thus, the CRC heterogeneity puzzle is far from being solved, and more research is necessary for prospective trials that separately investigate CC and RC. Our prospective study aimed to map the colon cancer landscape using 16S rRNA amplicon sequencing in biopsy samples from the terminal ileum, healthy colon tissue, healthy rectal tissue and tumor tissue as well as in preoperative and postoperative stool samples of 41 patients. While fecal samples provide a good approximation of the average gut microbiome composition, mucosal biopsies allow for detecting subtle variations in local microbial communities. In particular, the small bowel microbiome has remained poorly characterized, mainly because of sampling difficulties. Our analysis revealed the following: (i) right- and left-sided colon cancers harbor distinct and diverse microbiomes, (ii) the tumor microbiome leads to a more consistent cancer-defined microbiome between locations and reveals a tumor microbiome-ileal microbiome association, (iii) the stool only partly reflects the microbiome landscape in patients with CC, and (iv) mechanical bowel preparation and perioperative antibiotics together with surgery result in major changes in the stool microbiome, characterized by a significant increase in the abundance of potentially pathogenic bacteria, such as Enterococcus. Collectively, our results provide new and valuable insights into the complex microbiome landscape in patients with colon cancer.

Evaluation of FAM19A4/miR124-2 methylation performance in the management of CIN3 diagnosed pregnant women.

Pregnant women diagnosed with CIN3 have high regression rates after delivery. Biomarkers are needed to only identify pregnant women with progressive CIN requiring treatment to reduce overreferral and overtreatment. In our study we evaluated the performance of the FAM19A4/miR124-2 methylation test for molecular triage on FFPE samples of CIN3+-diagnosed pregnant women with known clinical course over time as well in a cross-sectional setting. In this German multicenter retrospective study biopsy material was collected from pregnant women diagnosed with cervical cancer (n = 16), with CIN3 that progressed to cancer during pregnancy (n = 7), with CIN3 that regressed to CIN1 or less within 6 months after delivery (n = 41), without CIN (n = 16), CIN3 covering 3-4 quadrants (n = 14) and randomly selected CIN3 (n = 41). FAM19A4/miR124-2 methylation analysis was performed blinded on first diagnosis. All pregnant women with cervical cancer and with CIN3 progressing to cancer tested positive for FAM19A4/miR124-2 methylation (100%, 22/22). In the regressing CIN3 group 47.5% and in the group without CIN 21.6% tested methylation positive. High-volume CIN3 and random selected CIN3 were methylation-positive in 91.7% and 82.1%, respectively. Methylation levels were significantly higher in progressive CIN3 and cancer compared to the controls (P < .0005). The likelihood ratio of a negative methylation test (LR-) for progressive CIN3+ was 0 (95% CI: 0-0.208). A negative FAM19A4/miR124-2 methylation test can rule out progressive CIN disease in pregnant women diagnosed with CIN3. This can help the clinician by managing these pregnant women with conservative follow-up until after delivery.

pT3 colorectal cancer revisited: a multicentric study on the histological depth of invasion in more than 1000 pT3 carcinomas-proposal for a new pT3a/pT3b subclassification.

BACKGROUND: Pathological TNM staging (pTNM) is the strongest prognosticator in colorectal carcinoma (CRC) and the foundation of its post-operative clinical management. Tumours that invade pericolic/perirectal adipose tissue generally fall into the pT3 category without further subdivision. METHODS: The histological depth of invasion into the pericolic/perirectal fat was digitally and conventionally measured in a training cohort of 950 CRCs (Munich). We biostatistically calculated the optimal cut-off to stratify pT3 CRCs into novel pT3a (≤3 mm)/pT3b (>3 mm) subgroups, which were then validated in two independent cohorts (447 CRCs, Bayreuth/542 CRCs, Mainz). RESULTS: Compared to pT3a tumours, pT3b CRCs showed significantly worse disease-specific survival, including in pN0 vs pN+ and colonic vs. rectal cancers (DSS: P < 0.001, respectively, pooled analysis of all cohorts). Furthermore, the pT3a/pT3b subclassification remained an independent predictor of survival in multivariate analyses (e.g. DSS: P < 0.001, hazard ratio: 4.41 for pT3b, pooled analysis of all cohorts). While pT2/pT3a CRCs showed similar survival characteristics, pT3b cancers remained a distinct subgroup with dismal survival. DISCUSSION: The delineation of pT3a/pT3b subcategories of CRC based on the histological depth of adipose tissue invasion adds valuable prognostic information to the current pT3 classification and implementation into current staging practices of CRC should be considered.

The impact of body mass index on prognosis in patients with colon carcinoma.

BACKGROUND: The impact of body mass index (BMI) on prognosis in patients with curatively resected stage I-III colon carcinoma was analyzed. METHODS: The prospectively collected data of 694 patients who underwent complete mesocolic excision between 2003 and 2014 were analyzed. BMI was classified into four categories: underweight (BMI < 18.5 kg/m2; n = 13), normal weight (BMI 18.5 to 24.9 kg/m2; n = 221), overweight (BMI 25.0 to 29.9 kg/m2; n = 309), and obese (BMI ≥ 30.0 kg/m2; n = 151). Univariate and multivariate analyses for comparison of prognosis were performed. RESULTS: The 5-year rate of locoregional recurrence in all 694 patients was 2.1%, and no differences were found with respect to BMI (p = 0.759). For distant metastasis, the 5-year rate for all patients was 13.4%, and BMI did not have a significant impact (p = 0.593). The 5-year rate of disease-free survival for all 694 patients was 72.4%. The differences with respect to BMI were not found to be significant in univariate analysis (p = 0.222). In multivariate Cox regression analysis, disease-free survival was significantly better in obese patients (HR 0.7; p = 0.034). Regarding overall survival, the 5-year rate for all patients was 78.1%. In univariate analyses, no significant differences were found for BMI (p = 0.094). In the Cox regression analysis, overweight and obese patients had significantly better survival (overweight: HR 0.7; p = 0.027; obese: HR 0.6; p = 0.019). CONCLUSION: The better survival of overweight and obese patients in multivariate analyses must be interpreted with caution. It is influenced by several factors and seems to correspond to the phenomenon of the obesity paradox.

Factors influencing downstaging after neoadjuvant long-course chemoradiotherapy in rectal carcinoma.

PURPOSE: This single-centre cohort study was designed to identify factors that can predict primary tumour downstaging by neoadjuvant chemoradiotherapy (nCRT) in rectal carcinoma. METHODS: Prospectively collected data from 555 patients with clinical T category (cT) cT3-4 rectal carcinoma treated between 1995 and 2019 were retrospectively analysed. All patients received long-term neoadjuvant chemoradiotherapy followed by surgery with curative intent at the Department of Surgery, University Hospital Erlangen, Germany. Patient-, tumour- and treatment-related factors with a potential impact on the downstaging of rectal carcinoma to pathological T category (pT) ≤ ypT2 and ypT0 were analysed in univariate and multivariate logistic regression analyses. The prognosis of patients with and without downstaging of the primary tumour was compared. RESULTS: A total of 288 (51.9%) patients showed downstaging to ≤ ypT2. Eighty-six (15.5%) patients achieved clinical complete regression (ypT0). In the multivariate logistic regression analysis, the factors cT category, BMI, ECOG score, CEA, histological type, extension in the rectum and year of the start of treatment were found to be independent factors for predicting downstaging to ≤ ypT2 after neoadjuvant chemoradiotherapy. The year of treatment initiation also remained an independent significant predictor for pathological complete regression. The prognosis was superior in patients with downstaging to ≤ ypT2 in terms of locoregional and distant recurrence as well as disease-free and overall survival. CONCLUSION: Factors predicting downstaging after long-term nCRT could be identified. This may be helpful for counselling patients and selecting the optimal treatment for patients with advanced rectal carcinoma.

Mucosal Mast Cell Distribution in the Gastrointestinal Tract of Children: A Preliminary Study for Establishing Reference Values.

OBJECTIVES: The physiological number and distribution of mast cells (MCs) in the pediatric gastrointestinal (GI) tract is not well defined and reference values of normality are missing. To define a physiological and disease defining cut-off, a systematic histological exploration of MC distribution from the esophagus to the rectum in healthy as well as in patients with gastrointestinal food allergies (GFA) was performed. METHODS: Nine pediatric subjects that exhibited unremarkable histopathological evaluations or underwent endoscopy for surveillance reasons after a previous polypectomy of single colonic juvenile polyps served as reference cohort. In all of these subjects, a chronic inflammatory disease (eg, inflammatory bowel disease, celiac disease) or allergy was excluded. In addition, a group of 15 patients with gastrointestinal complaints suspected to be caused by a GFA were investigated. Immunohistochemistry was performed from all biopsies using CD117 (c-Kit) as a reliable marker to identify MCs in the lamina propria. RESULTS: There were distinct differences of MC counts in all parts of the pediatric GI tract. The highest counts of MCs in both symptomatic patients and control cohort, were found in the duodenum, terminal ileum, cecum and ascending colon. The lowest counts were found in the esophagus. Significant disparities between GFA and healthy subjects were found in the gastric corpus (22.1 ±â€Š4.0/ high power field [HPF] vs 32.0 ±â€Š10.1/HPF; P = 0.034) and ascending colon (44.8 ±â€Š10.4/HPF vs 60.4 ±â€Š24.3/HPF; P = 0.047). CONCLUSIONS: Mucosal MC counts in the pediatric GI tract are higher than previously reported, with a considerable overlap between healthy and GFA patients. These results provide detailed information on distribution and numbers of MCs in pediatric allergic patients while allowing estimates of physiological values in childhood for the first time. With regard to diagnostic procedures in GFA further laboratory parameters have to be integrated.

Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice.

Experimental in vivo data have recently shown complementary neuroprotective actions of rhEPO and growth hormone (rhGH) in a neonatal murine model of hypoxic brain injury. Here, we hypothesized that rhGH and rhEPO mediate stabilization of the blood−brain barrier (BBB) and regenerative vascular effects in hypoxic injury to the developing brain. Using an established model of neonatal hypoxia, neonatal mice (P7) were treated i.p. with rhGH (4000 µg/kg) or rhEPO (5000 IU/kg) 0/12/24 h after hypoxic exposure. After a regeneration period of 48 h or 7 d, cerebral mRNA expression of Vegf-A, its receptors and co-receptors, and selected tight junction proteins were determined using qRT-PCR and ELISA. Vessel structures were assessed by Pecam-1 and occludin (Ocln) IHC. While Vegf-A expression increased significantly with rhGH treatment (p < 0.01), expression of the Vegfr and TEK receptor tyrosine kinase (Tie-2) system remained unchanged. RhEPO increased Vegf-A (p < 0.05) and Angpt-2 (p < 0.05) expression. While hypoxia reduced the mean vessel area in the parietal cortex compared to controls (p < 0.05), rhGH and rhEPO prevented this reduction after 48 h of regeneration. Hypoxia significantly reduced the Ocln+ fraction of cortical vascular endothelial cells. Ocln signal intensity increased in the cortex in response to rhGH (p < 0.05) and in the cortex and hippocampus in response to rhEPO (p < 0.05). Our data indicate that rhGH and rhEPO have protective effects on hypoxia-induced BBB disruption and regenerative vascular effects during the post-hypoxic period in the developing brain.

Tumor Infiltration with CD20+CD73+ B Cells Correlates with Better Outcome in Colorectal Cancer.

Immunotherapy has become increasingly important in the treatment of colorectal cancer (CRC). Currently, CD73, also known as ecto-5'-nucleotidase (NT5E), has gained considerable interest as a potential therapeutic target. CD73 is one of the key enzymes catalyzing the conversion of extracellular ATP into adenosine, which in turn exerts potent immune suppressive effects. However, the role of CD73 expression on various cell types within the CRC tumor microenvironment remains unresolved. The expression of CD73 on various cell types has been described recently, but the role of CD73 on B-cells in CRC remains unclear. Therefore, we analyzed CD73 on B-cells, especially on tumor-infiltrating B-cells, in paired tumor and adjacent normal tissue samples from 62 eligible CRC patients. The highest expression of CD73 on tumor-infiltrating B-cells was identified on class-switched memory B-cells, followed by naive B-cells, whereas no CD73 expression was observed on plasmablasts. Clinicopathological correlation analysis revealed that higher CD73+ B-cells infiltration in the CRC tumors was associated with better overall survival. Moreover, metastasized patients showed a significantly decreased number of tumor-infiltrating CD73+ B-cells. Finally, neoadjuvant therapy correlated with reduced CD73+ B-cell numbers and CD73 expression on B-cells in the CRC tumors. As promising new immune therapies are being developed, the role of CD73+ B-cells and their subsets in the development of colorectal cancer should be further explored to find new therapeutic options.

Prognostic impact of molecular muscle-invasive bladder cancer subtyping approaches and correlations with variant histology in a population-based mono-institutional cystectomy cohort.

PURPOSE: Recently discovered molecular classifications for urothelial bladder cancer appeared to be promising prognostic and predictive biomarkers. The present study was conducted to evaluate the prognostic impact of molecular subtypes assessed by two different methodologies (gene and protein expression), to compare these two approaches and to correlate molecular with histological subtypes in a consecutively collected, mono-institutional muscle-invasive bladder cancer (MIBC) cohort. METHODS: 193 MIBC were pathologically re-evaluated and molecular subtypes were assessed on mRNA (NanoString technology, modified 21-gene-containing MDACC approach) and protein levels (immuno-histochemical [IHC] analysis of CK5, CK14, CD44, CK20, GATA3 and FOXA1). Descriptive statistical methods and uni-/multi-variable survival models were employed to analyze derived data. RESULTS: Neither gene expression nor protein-based subtyping showed significant associations with disease-specific (DSS) or recurrence-free survival (RFS). Agreement between mRNA (reference) and protein-based subtyping amounted 68.6% for basal, 76.1% for luminal and 50.0% for double-negative tumors. Histological subtypes associated with RFS in uni-variable (P = 0.03), but not in multivariable survival analyses. Tumors with variant histology predominantly showed luminal subtypes (gene expression subtyping: 36/55 cases, 65.5%; protein subtyping: 44/55 cases, 80.0%). Squamous differentiation significantly associated with basal subtypes (gene expression subtyping: 44/45 squamous cases, 97.8%; protein subtyping: 36/45 cases, 80.0%). CONCLUSION: In our consecutive cystectomy cohort, neither gene, protein expression-based subtyping, nor histological subtypes associated with DSS or RFS in multi-variably adjusted survival analyses. Application of a limited IHC subtyping marker panel showed high concordance of 83.9% with gene expression-based subtyping, thus underlining the utility for subtyping in pathological routine diagnostics. In addition, histological MIBC subtypes are strong indicators for intrinsic subtypes.

Human papilloma virus genotype distribution in women with premalignant or malignant lesions of the uterine cervix.

OBJECTIVE: Cervical cancer is caused by persistent infection with high-risk human papillomavirus (hrHPV). Cytology-based national screening programs have reduced the incidence and mortality of cervical cancer. Different hrHPV subtypes have different carcinogenic potentials. This study evaluated the distribution of different types of hrHPV relative to age in cervical cancer and its precursor lesions. METHODS: HPV testing was performed between November 2018 and February 2020 using the Abbott RealTime high-risk HPV assay on an Abbott m2000sp instrument. This assay separately detects HPV-16, HPV-18, and a pool of 12 additional hrHPV types (HPV-31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68). RESULTS: The study included 652 women with HPV samples and biopsies of the cervix or histology samples obtained during surgery. In all, 30.8% (95% CI, 27.3-34.6%) were HPV-negative. Among HPV-positive women, HPV-16, HPV-18, and "HPV other" types were found in 33.5, 4.4, and 49.4%, respectively. Cervical intraepithelial neoplasia (CIN) 3/high-grade squamous intraepithelial lesions (HSILs) in women ≤ 34 years were positive for HPV-16 in 54.5% of cases and in those ≥ 35 years in 45.4% of cases. Among women with cervical cancer, 75.8% were infected with HPV-16 or had coinfection with HPV-16 and "HPV other". CONCLUSIONS: HPV-16 is the most common type of hrHPV in HSIL + lesions. It is more common in women diagnosed with CIN 3/HSIL who are aged ≤ 35 and is decreasing with age. Therefore, women age ≥ 35 with persistent infection with this type of hrHPV need careful surveillance, as they are at high risk of progression to cervical cancer.

Human CD22-Transgenic, Primary Murine Lymphoma Challenges Immunotherapies in Organ-Specific Tumor Microenvironments.

Targeted immunotherapies have greatly changed treatment of patients with B cell malignancies. To further enhance immunotherapies, research increasingly focuses on the tumor microenvironment (TME), which differs considerably by organ site. However, immunocompetent mouse models of disease to study immunotherapies targeting human molecules within organ-specific TME are surprisingly rare. We developed a myc-driven, primary murine lymphoma model expressing a human-mouse chimeric CD22 (h/mCD22). Stable engraftment of three distinct h/mCD22+ lymphoma was established after subcutaneous and systemic injection. However, only systemic lymphoma showed immune infiltration that reflected human disease. In this model, myeloid cells supported lymphoma growth and showed a phenotype of myeloid-derived suppressor cells. The human CD22-targeted immunotoxin Moxetumomab was highly active against h/mCD22+ lymphoma and similarly reduced infiltration of bone marrow and spleen of all three models up to 90-fold while efficacy against lymphoma in lymph nodes varied substantially, highlighting relevance of organ-specific TME. As in human aggressive lymphoma, anti-PD-L1 as monotherapy was not efficient. However, anti-PD-L1 enhanced efficacy of Moxetumomab suggesting potential for future clinical application. The novel model system of h/mCD22+ lymphoma provides a unique platform to test targeted immunotherapies and may be amenable for other human B cell targets such as CD19 and CD20.